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Previous Article | Next Article 
Journal of Bacteriology, August 2005, p. 5166-5178, Vol. 187, No. 15
0021-9193/05/$08.00+0 doi:10.1128/JB.187.15.5166-5178.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
A- and Repression of the EE-Responsive phoB-PS+V Promoters during Pho Response
Amr Eldakak, and
F. Marion Hulett*
Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607
Received 6 January 2005/ Accepted 25 April 2005
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ABSTRACT |
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E was necessary and sufficient for PS expression in vitro. PS expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of E is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP
P) repressed PS in vitro via direct binding to the promoter, the first example of an EE-responsive promoter that is repressed by PhoPP. Whereas either PhoP or PhoPP in the presence of EA was sufficient to stimulate transcription from the phoB-PV promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoPP were required for PV promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during Pi-replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Different patterns of PhoP binding are observed at PhoP-activated and -repressed promoters (for details see references 3 and 21). Activated promoters contain a core binding region positioned on the coding strand roughly between positions –20 and –60 relative to the transcription start site that contains four repeated 6-bp consensus PhoP-binding sequences that form two-dimer binding sites where PhoP dimers bind cooperatively (13). Activated promoters often have secondary PhoP dimer binding sites either 3' or 5' of the core binding region that are required for full promoter activity (13, 31). At repressed promoters phosphorylated PhoP (PhoPP) binds to regions that overlap the transcription initiation site, and in some cases PhoPP oligomerizes along the DNA as far as 168 bp into the coding region. Repressed promoters with only one PhoP dimer binding site, not two PhoP dimer binding sites, require phosphorylation for PhoP binding (4, 28).
Alkaline phosphatases in B. subtilis are encoded by members of a multiple-gene family that is expressed differentially under phosphate starvation conditions and sporulation conditions (14, 22). Phosphate starvation-inducible (PSI) alkaline phosphatases require the phoPR gene products, whereas sporulation-inducible alkaline phosphatases are expressed independent of the Pi concentration or phoP and/or phoR gene products but rather require the products of at least three stage II sporulation genes, including spoIIAC (encoding the forespore-specific sigma factor, F, which regulates the E activation), spoIIE (a PP2C phosphatase required for the activation of F), and spoIIGB (encoding the mother cell specific sigma factor, E) (5, 9). Two major alkaline phosphatases, PhoB (formerly APase III) and PhoA (formerly APase IV) (19), account for approximately 98% of total alkaline phosphatase specific activity in response to phosphate starvation (6, 24). Other alkaline phosphatases account for less than 2% of the total alkaline phosphatase specific activity induced during phosphate starvation, including that of PhoD phosphodiesterase (13, 14).
phoB is among the members of both the E-controlled genes and the Pho regulon genes that have recently been identified using DNA microarray analysis (15, 32), reporter gene technology screening (37), and transcriptional profiling (16). These genes include phoB-ydhF (2, 15, 32), phoPR (15, 32), yhaX (15, 37), yhbH (15, 37), yycP (15, 32), and glnQ (15, 32), all of which were reported to be PSI in a phoPR-dependent manner and are potentially transcribed from E-dependent promoters, at least during sporulation induction. Inclusion of the phoPR promoter among these genes and operons is explained by evidence indicating that three of the six phoPR operon promoters (35; A. Puri and F. M. Hulett, unpublished data) require PhoPP for enhanced transcription. One of these promoters is an EE-responsive promoter, and two are EA promoters (35).
phoB promoter deletion analysis was previously performed using a number of nonisogenic B. subtilis strains that contained mutations in important regulatory genes, including abrB, spo0A, spo0B, sigF, spoIIE, sigE, phoR, phoP, and phoS (9, 20). These studies established that phoB gene transcription is initiated from two transcription start sites at two different promoters. A strong vegetative cell promoter (PV) was phosphate starvation inducible only in a phoP- and/or phoR-dependent manner and accounted for approximately 40% of the total alkaline phosphatase activity during phosphate starvation. A 50-fold-weaker sporulation promoter (PS) was expressed at stage II during phosphate-replete growth on modified Schaffer's sporulation medium; its expression accounted for approximately 45% of the total alkaline phosphatase specific activity during sporulation induction (6, 22, 24).
Sequence analysis of the phoB promoter region (see Fig. 4A) revealed the presence of a consensus –10 region (TATAAT) for A-dependent promoters upstream of the PV transcription start site, but no consensus –35 region (TTGACA) for A-dependent promoters was observed (9). A moderately conserved consensus –10 region (AATAACCA) and a –35 region (TTCTAAA) for E-dependent promoters (15) were observed upstream of the PS promoter transcription start site. The response regulator PhoP was reported to bind to the phoB promoter exclusively between the PV and PS transcriptional initiation sites (29), a region which contains four direct repeats (TT[C/A/T]A[C/T]A) of the conserved sequence for PhoP binding (13, 30).
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P, A, and/or E in transcriptional activation from the phoB promoters. |
MATERIALS AND METHODS |
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E protein (*E), the sigE gene lacking the region encoding the prosequence was PCR amplified using JH642 chromosomal DNA with primers FMH490 and FMH491 containing cleavage sites for NdeI and BamHI, respectively. The PCR-amplified fragments were cloned into plasmid pCR2.1, resulting in plasmid pCH201. The mutant sigE gene was then released from pCH201 by digestion with NdeI and BamHI and cloned into plasmid pET16b at the same sites to generate plasmid pCH202. The accuracy of all plasmid constructs was confirmed by DNA sequencing. Plasmid pCH202 was used for transformation of E. coli BL21(DE3)/pLysS, and representative transformants were selected for the chloramphenicol resistance phenotype. Enzyme assays. For determination of total alkaline phosphatase specific activity, 250 µl of an LPDM culture was added directly to 1 ml of the substrate, 0.1 M p-nitrophenyl phosphate in 1 M CHES (N-cyclohexyl-2-aminoethanesulfonic acid; pH 9.5), and the reaction rates were measured at an optical density at 420 nm (OD420). One unit of APase activity was equivalent to 1 µmol of p-nitrophenol released per min at 37°C. The APase specific activity was expressed in units per unit of OD540.
The ß-galactosidase (ß-Gal) specific activity was measured by the method of Ferrari et al. (17) using o-nitrophenyl-ß-D-galactopyranoside as the substrate. One unit of ß-Gal activity was equivalent to 0.33 nmol of o-nitrophenol released per min at 37°C. The ß-Gal specific activity was expressed in units per milligram of total cellular protein. The amount of B. subtilis JH642 total cellular protein was calculated as previously described by Eder et al. (14).
Overexpression and purification of proteins.
For overexpression of His10PhoP, His10*E, GST*PhoR, and His6A proteins, E. coli BL21(DE3)/pLysS was used as a host for plasmids pCH128, pCH201, pLS21, and pLC2, respectively (Table 1). Overexpression and purification of His-tagged and glutathione S-transferase (GST)-tagged proteins were performed as described previously (29). *PhoR is the soluble cytoplasmic portion of the PhoR protein. The His tags were cleaved by factor Xa treatment, whereas the GST tag was removed by thrombin cleavage (29). For preparation of the B. subtilis His6-tagged RNA polymerase (His6RNAP) core enzyme (E), B. subtilis strain MH5636 (38) was grown in 12 1-liter portions of LB medium. Cells were collected at the end of the exponential phase of growth. Cell lysis and RNAP core enzyme purification were performed as previously described (38).
Gel mobility shift assay.
For preparation of probes, the phoB-PS+V promoter fragment was amplified by PCR using B. subtilis JH642 genomic DNA as the template and primers FMH745 and FMH746, whereas the phoB-PS promoter fragment was PCR amplified using plasmid pPS2 as the template and primers FMH124 and FMH128 (FMH124 and FMH128 are homologous to DNA in pDH32 adjacent to the EcoRI and BamHI cloning sites, respectively.). 32P labeling was performed during PCR by incorporating [
-32P]dATP and/or [-32P]dGTP (0.2 µCi/µl) into the newly synthesized DNA strand in the presence of limited dATP and/or dGTP concentrations (20 µM, usually one-tenth the concentration of dCTP and dTTP). The 32P-labeled probes were ethanol precipitated and extracted from a 1.2% agarose gel using a gel extraction kit (QIAGEN), and the final concentration was adjusted to 0.1 µM in nuclease-free transcription buffer (NFTB) (see below). The binding reactions (8 µl) were performed in NFTB containing, in order of addition, 12 nM 32P-labeled DNA (15,000 cpm), 180 µM ATP, 0.18 µM *PhoR, and PhoP at concentrations ranging from 0.35 to 2.8 µM. The reaction mixtures were incubated at 37°C for 30 min and were subsequently loaded onto 5% nondenaturing polyacrylamide gels (acrylamide/bisacrylamide, 29:1), which had been prerun in Tris-borate-EDTA buffer at 100 V/10°C for 30 min, and electrophoresed for 2 h at 100 V/10°C. The free probe and shifted PhoPP-DNA complex on dried gels were visualized using a PhosphorImager (Molecular Dynamics).
In vitro transcription analysis.
All components used for in vitro transcription reactions were adjusted to the required concentrations by dilution in NFTB (Tris-HCl [pH 7.8], 10 mM; KCl, 50 mM; MgCl2, 5 mM; CaCl2, 1 mM; EDTA-Na2, 0.1 mM) containing 5% glycerol, unless indicated otherwise. To obtain the template DNA for in vitro transcription analysis, the full-length phoB-PS+V promoter region (positions –222 to 106 relative to the translation start codon at position 1 [see Fig. 4A]) was PCR amplified using JH642 chromosomal DNA and primers FMH745 and FMH746 (Table 1), and the ykoL promoter region (positions –106 to 151 relative to the transcription start site at position 1 [see Fig. 6A]) was PCR amplified using primers FMH769 and FMH768. The phoB-PS promoter region was obtained by PCR using plasmid pPS2 as the template and primers FMH124 and FMH128. Promoter DNA was extracted from a 1.2% agarose gel using a gel extraction kit (QIAGEN), ethanol precipitated, and resuspended in NFTB to a final concentration of 100 nM. To prepare RNAP holoenzyme, core RNAP was incubated with each factor on ice for 30 min. For each transcription reaction (final volume, 15 µl), template DNA (33 nM) was mixed with ATP (180 µM), *PhoR protein (0.18 µM), and PhoP protein at different concentrations (0.02 to 10 µM in twofold increments) and incubated at 37°C for 20 min. *PhoR and PhoP dilutions were prepared in phosphorylation buffer (HEPES [pH 8.0], 50 mM; KCl, 50 mM; MgCl2, 50 mM; 7% glycerol) prior to addition to the reaction mixtures. To produce various concentrations of PhoPP in the in vitro transcription reaction mixtures, PhoP phosphorylation was performed during the initial DNA binding step using *PhoR at a fixed concentration, 0.18 µM. After the binding reaction, the reconstituted RNAP holoenzyme (core enzyme concentration, 0.09 µM; factor concentration, 0.7 µM) was added to the reaction mixture; this was followed by addition of a nucleoside triphosphate mixture (180 µM each of ATP, GTP, and CTP, 18 µM UTP, 0.1 µM [-32P]UTP [3,000 Ci/mmol; 10 µCi/µl], and 1 U/µl RNasin RNase inhibitor in NFTB) and incubation at 37°C for another 30 min. In vitro transcription reactions were stopped by addition of 7.5 µl stop buffer (7 M urea, 100 mM EDTA-Na2, 0.05% xylene cyanol, 0.05% bromophenol blue, 5% glycerol), and the mixtures were heated at 75°C for 5 min. Ten microliters of each reaction mixture was loaded onto a sequencing gel (6% polyacrylamide [19:1], 8 M urea) and electrophoresed at 350 V for 1 h. The RNA transcripts on dried gels were visualized with a PhosphorImager (Molecular Dynamics) and subjected to quantitative analysis using the volume report function of the ImageQuant software, version 5.1, with corrections for background by subtraction of intensities below and above each specified band. The radiolabeled RNA marker was prepared according to the manufacturer's instructions (Novagen).
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Determination of inorganic phosphate concentration.
The extracellular inorganic phosphate concentrations were determined for LPDM cultures using the method of Ames and Dubin (1). For analysis, 300 µl of properly diluted culture supernatant (usually 10% in deionized H2O for an LPDM culture) was mixed with 700 µl of ascorbic acid reagent (10% ascorbic acid in H2O and 0.4 g of ammonium molybdate liter in 1 N H2SO4, mixed at a ratio of 1:6 [vol/vol]), and the reaction mixtures were incubated at 45°C for 20 min. The inorganic phosphate concentration (µM) was equivalent to the OD820 of the reaction mixture divided by the slope obtained from a Pi standard curve. Under our experimental conditions the slope of the Pi standard curve was 0.008 OD820 unit/µM.
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RESULTS |
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100 µM), and peaked at T5, when maximum expression levels of 6,000 ß-Gal units were detected. Expression from the PS promoter fusion was induced at T4, and the levels were >50-fold lower than the levels from the PV promoter; however, they failed to reach the maximum levels in the first 12 h of growth. No expression from the PV promoter fusion was observed in the phoPR mutant grown in LPDM (Fig. 2B), indicating that the PSI expression from the strong PV promoter is phoPR dependent. In addition, phoPR deletion affected the timing of induction from the weak PS promoter. Expression from the PS promoter in phoPR mutants, from either the PS or PS+V promoter fusions, exhibited a further 2- to 3-h lag compared to the wild-type strain. During growth of the three sigE mutant strains in LPDM (Fig. 2C), no expression was observed from the PS promoter fusion, indicating that the PSI expression from the PS promoter is sigE dependent. Like expression in the wild-type strains, expression from the PV promoter in the sigE mutants, from either the PV or PS+V promoter fusions, exhibited the strong induction pattern that was induced at T0 and peaked at T5 during phosphate starvation. This expression was 40% higher than that in the parental strains. Sporulation-inducible phoB expression is solely from the sigE-dependent PS promoter. During growth of wild-type, phoPR, or sigE mutant strains in SSG medium (Pi-replete conditions), no expression was observed from the strong PV promoter fusion (Fig. 2D through F), indicating that the sporulation-inducible expression of phoB is from the weak PS promoter only. Both the wild-type and phoPR mutant strains (Fig. 2D and E, respectively) induced expression from the PS promoter, either from the PS+V or the PS promoter fusions, at T4 and peaked at T6, when the maximum expression levels (<80 ß-Gal units) were detected. No expression from the PS promoter fusion was observed during growth of the sigE mutant strains in either SSG medium (Fig. 2F) or LPDM (Fig. 2C).
phoPR mutation affects the level and timing of expression from the PS promoter during phosphate limitation.
To determine the extent to which a mutation in phoPR affected the timing and inducing strength of the PS promoter under phosphate deficiency stress conditions, we compared the parental and phoPR mutant strains for growth and expression from the PS promoter fusion for 24 h in LPDM (Fig. 3). In the parental strain, expression from the PS promoter fusion was induced at T4 and peaked at T8 with a maximum expression of 110 ß-Gal units. Expression from the PS promoter fusion was delayed 2 to 3 h in the phoPR mutant strain, and the maximum expression level was reduced about 25%. In addition, the maximum expression levels detected from the PS promoter in LPDM (Fig. 3) were approximately twofold greater than the level detected in SSG medium (Fig. 2D), suggesting that medium composition also influences the strength of expression from the sigE-dependent PS promoter.
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E-responsive promoter whose transcription in vitro is inhibited by high PhoPP concentrations.
To determine if the role of PhoP-PhoR and the mother cell specific sigma factor (E) in transcription of the phoB-ydhF operon was direct or indirect, an in vitro transcription analysis was performed using a 325-bp linear DNA fragment containing the full-length phoB promoter (102 bp upstream of PS [position 1] and 142 bp downstream of PV [position 1]) (Fig. 4A). The expected transcript initiating from the PS transcription start site was 223 nucleotides long. As shown in Fig. 4B, when PhoP was not present in the in vitro transcription reaction mixtures (lanes 1 through 8), E-containing RNA polymerase (EE) was sufficient for transcription initiation from the phoB-PS promoter. No detectable PS transcripts were observed when either core RNA polymerase or E was not present in the in vitro transcription reaction mixtures (data not shown). Eight reaction mixtures (lanes 1 to 8) containing decreasing concentrations of core RNAP (0.18 to 0.01 µM) and increasing concentrations of E (0.09 to 0.18 µM) yielded similar concentrations of transcript (lanes 1 to 4). EE (molar ratio, 0.09:0.7) was used in the following studies. When PhoP was present in the in vitro transcription reaction mixtures at concentrations ranging from 0.02 µM to 2.8 µM (Fig. 4B, lanes 9 through 16), no significant effect on transcription from the E-dependent PS promoter was observed at PhoP concentrations of 0.35 µM (lanes 9 through 13), but concentrations of >0.7 µM resulted in a reduction in transcription. In the presence of *PhoR, PhoP concentrations from 0.02 µM to 2.8 µM (lanes 17 through 24) resulted in greatly reduced transcription from the PS promoter. Thus, PhoP was not required for transcription initiation from the PS promoter, but the presence of low concentrations of PhoPP or high concentrations of PhoP repressed transcription from the E-dependent PS promoter.
PhoPP and EA requirement for transcription from the phoB-PV promoter in vitro.
For in vitro transcription analysis of the PV promoter, we used the reaction conditions described above, except that we initiated transcription with A-saturated RNAP (EA). The expected transcript initiating from the PV transcription start site (position 1) was 142 nucleotides long. As shown in Fig. 4B (lower panel), in the absence of PhoP, A-containing RNA polymerase (EA) failed to stimulate transcription from the phoB promoter (lanes 1 through 8) at any concentration used. Addition of PhoP at concentrations ranging from 0.02 to 2.8 µM (lanes 9 through 16) stimulated a dose-dependent increase in transcription from the PV promoter only when the unphosphorylated PhoP concentration was 
0.18 µM (lanes 12 through 16). When *PhoR was present in the in vitro transcription reaction mixtures (lanes 17 through 24), PhoP concentrations ranging from 0.02 to 0.18 µM (lanes 17 through 20) stimulated a dose-dependent increase in transcription from the PV promoter, and the maximal yield was obtained at a PhoP concentration of 0.18 µM (lane 21). Further increases in PhoP concentrations (lanes 21 through 24) resulted in reduced transcription. The use of PhoP at concentrations of 1.4 µM significantly inhibited transcription activation from the PV promoter (lanes 23 and 24).
PhoP inhibition of PS promoter transcription is specific and involves PhoP binding to the PS promoter in addition to the PV promoter.
A phoB promoter fragment containing only the PS promoter (from pPS2 [Fig. 1]) was used as a template for in vitro transcription to determine if reduction of the PS transcript from the phoB-PS+V promoter in the presence of phosphorylated PhoP was exclusively the result of PhoP binding to the PV promoter core binding region, which blocked RNAP passage (Fig. 5A, top panel). Lane 1 contained the transcript from the EE-dependent PS promoter without PhoP or *PhoR. Lanes 2 through 6 contained increasing concentrations of PhoP (0.09 to 1.4 µM) plus *PhoR, which were shown to increasingly inhibit PS transcription from the phoB-PS+V promoter in the presence of *PhoR (0.18 µM) and ATP (Fig. 4B, upper panel). Under these conditions higher concentrations of PhoP increasingly inhibited the PS promoter, although not as strongly as identical concentrations did when the phoB-PS+V promoter was used as the template. A dominant role for PhoPP in PS inhibition was suggested by the strong transcript in the reaction with 2.8 µM PhoP in the absence of *PhoR (lane7). Quantitation of the PS transcript with increasing concentrations of PhoP plus *PhoR from the PS template alone is compared in Fig. 5A (bottom panel) to quantitation of the transcripts from the phoB-PS+V promoter fragment (Fig. 4B) with the same concentrations of PhoP or PhoP and *PhoR or with EE alone. Note the difference in PS transcription in the reaction mixture containing 1.4 µM PhoP with the full-length PS template in the presence of *PhoR.
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E-dependent promoter, spoIIID, under conditions described above (Fig. 5A, bottom panel) that inhibited the phoB-PS+V promoter. Lane 1 in Fig. 5B shows the level of transcript from the phoB-PS (top panel) or spoIIID (lower panel) template with EE alone. EE with PhoP (2.8 µM) resulted in some decrease in the PS promoter transcript, but in the presence of *PhoR (0.18 µM) and PhoP the transcript was completely inhibited, similar to the results obtained for the phoB-PS+V promoter (Fig. 4B, top panel, lane 24). Under the same conditions and when the *PhoR concentration was increased from 0.18 to 0.35 µM, the level of the spoIIID transcript (lanes 3 and 4, respectively) was similar to the level observed with EE without PhoP and *PhoR (lane 1), indicating that the role of PhoP and *PhoR in inhibition of the phoB promoter is specific.
Because transcription from the PS promoter was reduced in the presence of PhoPP, we asked if PhoP directly bound to the PS promoter fragment in a gel mobility shift assay and how the binding compared to PhoP binding to the full-length phoB promoter (Fig. 5C). Lane 1 in Fig. 5C contained the full-length phoB-PS+V free probe in the top panel and the PS free probe in the bottom panel. Lanes 2 to 8 in each panel contained increasing concentrations of PhoP in the presence of ATP and 0.18 µM *PhoR. Both probes revealed a partial shift at the lowest concentration of PhoP (lane 2), although the percentage of the phoB-PS+V probe shifted was significantly greater than the percentage of the PS probe shifted. Roughly twofold-higher concentrations of PhoP were required for a complete shift of the phoB-PS probe (1.4 µM PhoP) (Fig. 5C, bottom panel, lane 6) than for a complete shift of the phoB-PS+V probe (0.7 µM PhoP) (Fig. 5C, top panel, lane 4). These data indicate that PhoP binds directly to the PS promoter independent of the core binding region in the PV promoter previously reported. In light of the current data, we reexamined the PhoP footprinting protection on the phoB-PS+V promoter (29). While nearly complete protection of the large core binding region of the PV promoter was observed even at the lowest PhoPP concentration (55 nM), there was also a previously unnoticed, smaller protected region upstream of the Ps transcription start site on the noncoding strand. Partial protection was observed starting at 110 nM PhoPP, and there was increasing protection at PhoPP concentrations of 220 and 440 nM. The protected region in the promoter sequence is indicated in Fig. 4A. Interestingly, the protected region on the noncoding strand contains two putative consensus repeats for a PhoP dimer-binding sequence on either side of the –10 element of the EE-responsive PS promoter (–2TAAATAATGGTTATTCTTT–19), with a conservation match of 5/6 and 4/6, respectively. PhoP protection at this PS promoter site required PhoR and ATP for PhoP phosphorylation, just as retardation of the PS promoter probe in the studies described above did (data not shown).
PhoP activates in vitro transcription from the phoB-PV promoter in a phosphorylation- and/or concentration-dependent manner.
The concentration range of PhoP used was expanded up to 10 µM to compare the maximum transcription of PV with PhoP compared to that with PhoPP and to determine if high concentrations of PhoP inhibited transcription from the PV promoter (Fig. 6A). Because these experiments were designed to investigate the effects of various unphosphorylated PhoP and PhoPP concentrations on transcription from the PV promoter, the only experimental variable was the PhoP concentration, and all other reaction components were unchanged. This strategy resulted in three changes in the ratio of PhoP to PhoR. In the first case, the concentration of PhoP (0.02 to 0.09 µM) was less than that of PhoR (0.18 µM). Consistent with the data shown in Fig. 4B, this concentration range of PhoP stimulated transcription from the PV promoter only when PhoR was present (Fig. 6A). In the second case, PhoP and PhoR were present at equimolar concentrations or nearly equimolar concentrations. The concentration of PhoPP (0.18 µM) was the optimum concentration for maximum transcriptional yield from the PV promoter. In the last case, PhoP concentrations between 0.35 and 10 µM were higher than the PhoR concentration. In contrast to the other cases, PhoP at concentrations of >0.18 µM in the presence of PhoR resulted in linearly decreasing transcriptional yields, and complete repression was detected at concentrations of 1.4 µM. Interestingly, the same range of PhoP concentrations was able to stimulate transcription from the PV promoter in the absence of PhoR. The optimum unphosphorylated PhoP concentration that stimulated the maximum transcriptional yield from the PV promoter was 2.8 µM or near 2.8 µM, a repressing concentration in the case of PhoPP. Decreased transcription from the PV promoter was also observed at higher concentrations of unphosphorylated PhoP (concentrations above 5.25 µM). Thus, both unphosphorylated PhoP and PhoPP stimulated transcription from the PV promoter in a concentration-dependent manner for a maximum transcriptional yield, and addition of higher concentrations of either form decreased transcription from the PV promoter. The maximum transcriptional yield with the unphosphorylated PhoP was 85% that obtained with PhoPP and required 16-fold-higher PhoP concentrations.
To determine if phosphorylation of PhoP might influence its interaction with the EA form of RNAP, we varied the concentration of A in the reconstituted RNAP (Fig. 6B) while PhoP and PhoPP were used at their optimal concentrations, determined as described above. At the optimal PhoPP concentration (0.18 µM), limiting concentrations of A (0.02 µM) stimulated transcription, although the transcriptional yield was reduced compared to that obtained using higher A concentrations. Increasing the A concentration so that it was up to eightfold greater than the PhoPP concentration had no significant effect on the transcriptional yield. In contrast, the A concentration significantly influenced the ability of the unphosphorylated PhoP to stimulate transcription from the PV promoter in vitro. Concentrations of A of <0.35 µM were not sufficient to allow the optimal unphosphorylated PhoP concentration to stimulate transcription. A concentrations of 0.35 µM allowed maximum transcription activation at the optimal unphosphorylated PhoP concentration. Interestingly, A was required at a 17-fold-higher concentration to allow transcription activation at the optimum PhoP concentration compared to the concentration required at the optimum PhoPP concentration.
In general, this in vitro transcription analysis illustrated two different conditions for PhoP-dependent transcription activation from the EA-responsive phoB-PV promoter. The first condition is phosphorylation dependent, which allows a relatively low PhoPP concentration compared to the PhoP concentration to stimulate transcription even at limiting A concentrations. The second condition is concentration dependent, requiring considerably higher concentrations of both PhoP and A in order to stimulate transcription in vitro.
Unphosphorylated PhoP or PhoPP is essential for transcriptional activation of a second PhoP-regulated promoter, ykoL.
Because the ability of unphosphorylated PhoP to activate transcription in vitro was observed for the first time during the course of this study, we explored the use of a second PhoP-regulated promoter. The ykoL promoter exhibits significant similarity to the phoB promoter (Fig. 7A). First, it is transcribed from a potential A-dependent promoter which also lacks a consensus –35 sequence essential for EA recognition. Second, it contains the four TT(C/A/T)A(C/T)A repeats characteristic of PhoP-activated promoters, which are quite similar to the phoB promoter in terms of conservation, relative distances between tandem sequences, and position upstream of the transcription start site (41). Recently, DNase I footprint analysis of the ykoL promoter region with PhoP indicated that the levels of protection afforded by the phosphorylated and unphosphorylated forms of PhoP were similar (36), a situation that we observed for the phoB-PV promoter. The in vitro transcription analysis of the ykoL promoter (Fig. 7B) showed that despite the ability of EA alone to direct transcription from a nonspecific start site (PX), which produced an RNA transcript that was 100 nucleotides long (Fig. 7B, lanes 2 through 8), no PV transcript was observed. Addition of PhoP in the absence of *PhoR (lanes 9 through 16) or in the presence of *PhoR (lanes 17 through 23) switched transcription initiation to a site that yielded a transcript that was 150 nucleotides long, consistent with the PV transcription start site that was identified previously by primer extension analysis of the ykoL mRNA during growth of a wild-type strain in phosphate starvation medium (41) (Fig. 7A). Similar to the maximum transcriptional yield with the phoB promoter, the maximum transcriptional yield observed with the ykoL-PV promoter in the presence of unphosphorylated PhoP was 75% that observed in the presence of PhoPP. Higher concentrations of unphosphorylated PhoP decreased transcription activation from the ykoL-PV promoter start site, and there was a 10% reduction in the transcription yield from the ykoL-PV start site at a PhoP concentration of 10 µM (lane 16). PhoPP (0.35 µM) showed maximal transcription activation. Figure 7C shows quantitation of the ykoL-PV transcript with PhoP alone (lanes 9 to 16) and with PhoP plus *PhoR (lanes 17 to 23).
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1 µM (Fig. 8B) during the pre-Pho induction period (from T–2 to T0). When the Pi concentration in the medium decreased below 100 µM, alkaline phosphatases were induced and PhoP concentrations began to increase. Total APase specific activity increased rapidly until T2, while the PhoP concentrations continued to increase until T4. At T4, the PhoP concentration was approximately threefold higher than the concentration detected at T0. These data are consistent with our understanding of the two APase promoters and the complex phoPR promoter. It is likely that production of PhoP continues into the stationary phase because the phoPR operon has equally strong, PhoPP-enhanced EA and EE promoters. In contrast, the APase genes each have a strong, PhoP-dependent EA promoter (phoA and phoB-PV), but the EE promoter of phoB-PS is very weak (roughly 2% the strength of the EA promoter) and it appears to be inhibited by PhoPP at high concentrations. Thus, the contribution of the weak EE phoB-PS promoter to total APase activity should be negligible, like its contribution to phoB expression from the phoB-PS+V promoters during phosphate starvation (Fig. 2A).
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DISCUSSION |
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E-responsive phoB-PS promoter.
In vitro transcription data reported here established that EE was necessary and sufficient for phoB-PS promoter function. A direct role for EE in phoB-PS promoter expression was corroborated by phoB-PS promoter fusion data obtained with a sigE mutant strain during phosphate-limited growth in LPDM and during sporulation in SSG medium (Fig. 2). Previous phoB-lacZ fusion data (9) failed to identify PS promoter expression during phosphate-limited growth because the promoter fusion contained the 50-fold-stronger PV promoter in addition to the PS promoter, which masked the contribution of the PS promoter. PS promoter expression during phosphate-limited growth was delayed 2 h in the phoPR mutant (Fig. 3), suggesting that either synthesis or maturation of E is delayed under the Pi deficiency stress conditions in the absence of PhoPR. This implies that PhoPR or another Pho regulon gene is needed for efficient sporulation, which may be especially important in nature as inorganic phosphate is often the most limiting nutrient in the soil (34). PS promoter expression from the PS+V promoter fusion was not observed previously in phoP or phoR mutant strains grown in LPDM because experiments were terminated before delayed PS promoter activation at T5 to T6 (9).
PhoP and PhoR have two roles in transcription from the phoB-PS promoter. As stated above, the positive role for phoPR affects the timely expression from the EE-responsive promoter, PS, during phosphate starvation. At the same time, our in vitro data suggest that PhoPP represses transcription from the PS promoter by binding directly to the PS promoter. These results suggest that PhoPR has dual roles in development affecting the timing of at least one EE promoter by positively affecting when EE is available during phosphate starvation and also preventing early expression via PhoPP before Pho regulation is turned off. The fact that the latter role was not detected during previous induction studies of PS in phosphate-replete sporulation media when the PV promoter was silent is understandable because there would have been no signal for PhoP phosphorylation and thus no repression of PS (9).
Although the binding site in the phoB-PS promoter, upstream of the PV promoter, is positioned like other secondary binding sites for PhoP-activated promoters, it is clear that it has no role in PV activation because expression from the phoB-PS+V promoter fusion and expression from the PV promoter are the same (Fig. 2A and C). A requirement for phosphorylation of PhoP for binding at PS was observed for two other PhoP repressed promoters, resD and tagA (4, 28), that contain a single consensus PhoP dimer binding site.
Different mechanisms control genes and operons that have been classified as both Pho and EE regulon members.
For genes classified as both Pho and EE regulon genes, the mechanism responsible for inclusion of the phoPR promoter has been explained by PhoPP amplification of A- and E-responsive phoPR operon promoters (35). Here we showed that the PhoP-regulated phoB promoter, PV, is a A promoter and that transcription from the second promoter, PS, requires only EE, although the PS transcription was delayed in a phoPR mutant (Fig. 2B and 3) and was negatively regulated by PhoPP. yhaX and yhbH are similar to the phoB-PS promoter in three ways. Both these genes were expressed during Pi-limiting growth in a wild-type phoPR strain at a time later than the time that other Pho-regulated genes were expressed, neither gene was transcribed in a sigE mutant strain, and like the PS promoter, neither yhaX nor yhbH has consensus tandem repeats for PhoP binding on the coding strand upstream of the putative EE promoter (37). Because the Pho regulon assignment for yhaX and yhbH was based solely on the absence of promoter expression in a phoR mutant strain, it seems that the possible delay of EE-dependent gene expression in that strain, not the lack of direct PhoPR promoter regulation, may have caused the negative results. Consistent with this idea, PhoP does not bind to the yhaX promoter (36). No further information is available on the mechanism of Pho regulation and E-dependent regulation of yycP or glnQ. The putative yycOP and glnQ promoters are interesting in that both contain two putative consensus repeats for a PhoP dimer binding site on the coding strand on either side of the –35 element of the putative E promoter. It should be interesting to determine if EE-responsive PhoP-activated genes exist (in addition to autoregulation) and if their requirement for PhoP binding is different than the requirement for all PhoP-activated A promoters studied (13) or for the PhoP-enhanced autoregulation EE promoter (35).
PhoP or PhoPP plus EA is sufficient for phoB-PV or ykoL promoter transcription in vitro, and the PhoP concentration is the key.
The failure of EA to activate in vitro transcription of phoB-PV (Fig. 4) or ykoL (Fig. 7) is likely due to the absence of a –35 A consensus binding region in either promoter, as is true for other EA PhoP-activated promoters which absolutely require PhoPP for activation, including the phoA, pstS, and tuaA promoters (30, 40). Previous studies indicated that unphosphorylated PhoP was unable to activate transcription in vitro. The maximum concentrations of unphosphorylated PhoP and A used in the previous studies were 0.25 µM and 0.02 µM, respectively. Consistent with the data presented here (Fig. 5B), these concentrations of unphosphorylated PhoP plus EA were not sufficient to activate transcription in vitro. Here, we showed that unphosphorylated PhoP stimulated transcription from the phoB-PV promoter in vitro. However, the concentration of unphosphorylated PhoP required for maximum transcription was much higher than that required for PhoPP with either promoter (17-fold higher for phoB and 10-fold higher for ykoL), indicating that phosphorylation has an important role.
While it is possible that PhoP isolated from E. coli is phosphorylated, the activity of equimolar concentrations of PhoP compared to the activity of PhoP in the presence of PhoR plus ATP suggests that the percentages of PhoP phosphorylated in the two cases differ considerably. Furthermore, a PhoPN structural analysis (3) for crystals formed in 1 day at 4°C showed no phosphorylation. However, a number of E. coli-expressed unphosphorylated response regulators have been shown to activate transcription in vitro (PrrA [10], Spo0A [27], and DesR [11]), only to lose this ability in the variant with a mutation at the phosphorylatable aspartate residue and in strains containing this variant (PrrA [10] and DesR [11]). Spo0A (26) and DesR (11) were proven to be phosphorylated when they were isolated. UhpA or the UhpA D54N variant is constitutively active for expression of the sugar phosphate transporter UhpT in E. coli when it is overexpressed, but the variant is totally inactive as a single copy, suggesting that phosphorylation of UhpA is required for a single copy but not when UhpA is overexpressed (12, 33, 47). In the case of the PhoP/PhoQ two-component system of Salmonella, overexpression of the response regulator PhoP or the D52A variant activated regulon genes in vivo, albeit in a signal-independent manner, reportedly due to concentration-dependent activation of PhoP via dimerization. Further studies indicated that signal-dependent (low-Mg2+) PhoP DNA binding is phosphorylation dependent in vivo (46). In contrast to the examples described above, ResD D57A was shown to induce transcription of the ResDE regulon in response to oxygen limitation, suggesting that ResD senses oxygen-limiting conditions via an unknown mechanism in addition to being activated by phosphorylation by ResE (18).
Because of the high concentrations of unphosphorylated PhoP required for Pho regulon promoter activation in vitro and the modest threefold autoinduction of PhoP in vivo, we believe that a physiological role for unphosphorylated PhoP in a wild-type strain is unlikely. However, activation of Pho regulon genes has been reported in a phoR deletion strain. The ability to induce low-level expression from both tuaA and pstS promoters was retained in a phoR mutant strain during phosphate-deficient growth (30, 40). In the latter study (40), the authors proposed that PhoP was likely phosphorylated in a PhoR-independent manner (i.e., by unknown phosphodonors). In a second case, Ogura and colleagues (32) reported that overproduction of PhoP in vivo in a phoR deletion strain activated expression of phoB and a number of Pho regulon genes in a signal-independent manner; these genes are not normally activated in a phoR mutant during phosphate starvation. Although a number of possibilities may explain this observation, during PhoP overproduction under nonphysiological conditions concentration-dependent activation of PhoP is a potential factor.
Phosphorylation of PhoP minimizes the EA levels required for maximum transcription activation in vitro, consistent with in vivo conditions during Pho induction.
Because both unphosphorylated PhoP and PhoPP are dimers in solution (29) and both forms of PhoP bind to phoB (29) or ykoL (36) promoters with similar affinity, we asked if phosphorylation of PhoP had an effect on its interaction with the transcriptional machinery (7). Consistent with this notion, when PhoP or PhoPP was used at the concentration determined to be optimal for phoB-PV transcription (Fig. 5A), concentrations of A (0.09 µM) required for PhoPP-stimulated transcription activity were significantly lower than the concentrations required by unphosphorylated PhoP (Fig. 5B). Analysis of the in vivo concentration of PhoP showed that autoinduction of PhoP synthesis and APase expression initiated in parallel, when PhoP levels were the lowest, at T0. During the following transition period (T1 and T2), PhoP concentrations increased modestly, roughly onefold, while the abundance of EA is known to decrease due to sigma factor displacement (23). Taken together, these data suggest that the phosphorylated form of PhoP is probably responsible for in vivo activation of Pho regulon genes, including genes encoding APases.
Elevated concentrations of PhoPP inhibited transcription in vitro, providing a possible explanation for previous in vivo data.
PhoPP is known to function not only as a transcription activator but also as a transcription repressor of at least three EA-responsive promoters, including tagA/D (30, 39) and resD (4). One difference observed in vitro between repressed and activated promoters is the customary requirement for PhoP phosphorylation for promoter binding at PhoP-repressed promoters (4, 30) compared to activated promoters, where either PhoP or PhoPP binds. Also, it appears that similar levels of PhoPP are required for activation and repression in vivo because repression of one set of genes and activation of another set by PhoPP occur simultaneously. For example, upon phosphate starvation the genes responsible for synthesis of teichoic acid (the tagA/D operons) are repressed (30), while the tuaA operon encoding proteins for the synthesis of teichuronic acid is activated (30), both in a phoPR-dependent manner. Thus, it seems unlikely that elevated levels of PhoPP have a physiologically relevant role in Pho gene repression in a wild-type strain. However, when overexpression studies are conducted, PhoPP may play a role. Data consistent with in vivo repression of Pho regulon genes by elevated PhoPP concentrations come from two in vivo studies, both involving nonphysiological overexpression of PhoP. It was observed that expression of PhoP from its own promoter on a multicopy plasmid in a phoPR wild-type background resulted in a reduced growth rate and repression of Pho regulon genes even under Pi-deficient conditions (W. Liu and F. M. Hulett, unpublished). In an independent study (32), overproduction of any one of three response regulators (PhoP, DegU, and ComA) in a wild-type background failed to result in expression of target genes, and in each case deletion of a cognate histidine kinase gene was required for expression. Our in vitro data are consistent with the hypothesis that repression by elevated concentrations of PhoPP is one possible explanation for the previously observed in vivo repression of Pho regulon genes.
In summary, data presented here show that phoB-PS is an EE-responsive promoter that is expressed under phosphate-deficient conditions and that the timing of this expression is delayed in a phoPR mutant strain. These observations suggest that workers should be cautious when analyzing promoters believed to depend on both EE and phoPR. While a role for PhoPP in activation of an EE promoter has been reported previously (35), repression of the PS promoter by PhoPP is the first example of a repressor role for PhoPP at EE-responsive promoters. Two EA promoters that require PhoP for expression in vivo and in vitro, phoB-PV and ykoL, were shown to be activated at a high concentration of PhoP and to be repressed by high concentrations of PhoPP in vitro, thereby providing a possible explanation for previous in vivo observations (32, 39). Phosphorylation of PhoP resulted in decreased concentration requirements for both PhoPP and EA for stimulation of Pho regulon promoters in vitro.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant GM-33471 to F.M.H.
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FOOTNOTES |
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