GENETICS AND MOLECULAR BIOLOGY

Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of theEscherichia coli Phosphate Regulon

Andreas Haldimann, Larry L. Daniels, Barry L. Wanner
DOI: 

ABSTRACT

Escherichia coli genes regulated by environmental inorganic phosphate (Pi) levels form the phosphate (Pho) regulon. This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the Pi-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU. Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated ParaB promoter or the rhamnose-regulated PrhaB promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of ParaB orPrhaB fusions and for recombination of them onto the E. coli chromosome at the araCBAD orrhaRSBAD locus, respectively. Using strains carrying such single-copy fusions, we showed that a PrhaB fusion is more tightly regulated than a ParaB fusion in that a PrhaB-phoR + fusion but not a ParaB-phoR + fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, bothParaB-phoB + andPrhaB-phoB + fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action. We also used these fusions to study PhoU. Previously, we had constructed strains with precise ΔphoU mutations. However, we unexpectedly found that such ΔphoU mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol. 175:6797–6809, 1993). They also readily give rise to compensatory mutants with lesions inphoB, phoR, or a pst gene, making their study particularly difficult. Here we found that, by usingParaB-phoB +,PrhaB-phoB +, orPrhaB-phoR + fusions, we were able to overcome the extremely deleterious growth defect of a Pst+ ΔphoU mutant. The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.

The control of the Escherichia coli phosphate (Pho) regulon by environmental inorganic phosphate (Pi) levels is a paradigm of a bacterial signal transduction pathway in which occupancy of a cell surface receptor (the Pi-specific binding protein PstS) regulates gene expression in the cytoplasm (reference 29 and references therein). This signal transduction pathway requires seven proteins, all of which probably interact in a membrane-associated signaling complex. The Pi signaling proteins include (i) two members of the large family of two-component regulatory systems, namely, response regulator PhoB (a transcriptional activator) and its partner, histidine sensor kinase PhoR (itself an integral-membrane protein); (ii) four components of the ATP-binding cassette family Pi-specific transport (Pst) machinery (PstA, PstB, PstC, and PstS); and (iii) a negative regulator of unknown function called PhoU.

We proposed elsewhere that the Pi signaling response involves three processes: activation, deactivation, and inhibition (30). Accordingly, activation occurs under conditions of Pi limitation and requires both PhoB and PhoR; activation involves autophosphorylation of PhoR by ATP, phosphotransfer to PhoB, and transcriptional activation of Pho regulon promoters by phospho-PhoB (P-PhoB). Deactivation is a distinct intermediate step-down process that occurs upon a growth shift from Pi-limiting to Pi excess conditions. It is required to reestablish inhibition and leads to the dephosphorylation of P-PhoB in a process requiring PhoR and an excess of either PhoU, a Pst component(s), or both. Inhibition prevents phosphorylation of PhoB when Piis in excess; it requires all seven Pi signaling proteins (PhoB, PhoR, PhoU, PstA, PstB, PstC, and PstS) in an “inhibition complex” that, by insulating PhoB, interferes with its phosphorylation.

In order to study how PhoU and the Pst system interact with PhoB and PhoR, we had previously constructed mutants with defined deletions of the pstSCAB-phoU operon. This led to our discovery that a ΔphoU (unlike a phoU missense) mutation causes a severe growth defect, resulting from an apparent Pisensitivity phenotype (26). Growth inhibition by Pi has also been seen in mutants of the Pst transporter in which the Pi transport channel is permanently “switched on” (open), although in that case the growth defect is much less severe (34). The deleterious growth phenotype resulting from a ΔphoU mutation also leads to the accumulation of compensatory mutants with lesions in phoB, phoR, or a pst gene under normal growth conditions (26). Indeed, the poor growth of a ΔphoU mutant was apparently responsible for another laboratory concluding incorrectly that a ΔphoU mutation abolishes Piuptake (19), as their “ΔphoU mutant” carries also a linked pst mutation (29). On the contrary, we proved that a ΔphoU mutation has no effect on Pi uptake (26).

We have now found ways to overcome the difficulties of studyingphoU and (presumably) open-channel pst mutations as well. The growth defect due to a ΔphoU mutation is apparent only when the Pst system is synthesized at a high level, which in turn requires increased amounts of PhoB and PhoR, whose synthesis is autogenously controlled (10). We uncoupled phoBexpression and phoR expression from their normal controls by placing them behind the foreign, arabinose-regulatedParaB or rhamnose-regulatedPrhaB promoter. Upon induction of the corresponding ΔphoB or ΔphoR mutant with arabinose or rhamnose, such strains show nearly normal Picontrol of the Pho regulon. However, the fold induction is lowered. Importantly, a ΔphoU mutation has no deleterious effect on growth of these strains, even in the presence of the respective inducer. Our methods for constructing and characterizingParaB and PrhaB fusions may also be generally useful.

MATERIALS AND METHODS

Media and culture conditions.Luria-Bertani broth, tryptone-yeast extract, and M63 were routinely used as complex and minimal media. These were prepared as described elsewhere (28). To maintain plasmids, antibiotics (Sigma, St. Louis, Mo.) were added as follows: ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 50 μg/ml, and tetracycline at 12.5 μg/ml. Recombinants with a single-copy plasmid were selected with gentamicin at 4 μg/ml or kanamycin at 10 μg/ml. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal; Bachem, Torrance, Calif.) was used at 40 μg/ml to detect β-galactosidase. Tetracycline-sensitive (Tets) cells were selected on tetracycline-sensitive-selective (TSS) agar as described elsewhere (17). MacConkey agar (Difco, Detroit, Mich.) containing 1% l-arabinose, lactose, orl-rhamnose (Sigma) was used to test for the use of these carbon sources. Ara− and Rha− strains were verified by their inability to grow on M63 media containing 0.2%l-arabinose or l-rhamnose, respectively. Ara− and Rha− recombinants are distinguishable from arabinose- or rhamnose-sensitive ones by the inability of the latter to grow on MacConkey or glycerol agar in the presence of arabinose or rhamnose, respectively. In contrast, Ara− and Rha− recombinants are insensitive to these carbohydrates.

MOPS (morpholinepropanesulfonic acid) media containing different carbon sources were used to study gene regulation inParaB , PrhaB , andPrhaS fusion strains. Cells were grown on MOPS agar containing the same carbon source at 0.2% ford-glucose, d-fructose, andd-mannitol or 0.4% for glycerol, but without an inducer. Fresh isolated colonies (after less than 20 h of growth) were used to inoculate 0.06% glucose–, fructose–, or mannitol–MOPS medium or 0.1% glycerol–MOPS medium without or with an inducer. Cultures were incubated at 37°C for 16 to 24 h prior to the assay. Such carbon-limited cultures yield highly reproducible values that are qualitatively similar to ones obtained for logarithmic-growth phase cultures (27). l-Arabinose,l-rhamnose, and isopropyl-β-d-galactopyranoside (IPTG; Sigma) were used at 1.3, 1.1, and 0.2 mM, respectively, for induction.

Bacteria.All strains assayed are described in Table1. Others included BT333 (endA::tetAR; from W. Wackernagel [4]), BW5045 (srlC300::Tn10; [18]), BW8078 (λrecA + recA1; [28]), BW21391 (leu-63::Tn10; [12]), BW22773 (lacI q rrnB T14ΔlacZ WJ16 proC::Tn5-132; [14]), CA10 (galU95; from M. Berlyn), JW383 (metF159 zii-510::Tn10 thi-1; from M. Berlyn), W3110trpB114 (trpB114::Tn10; from C. Yanofsky), and ZK1001 (cysC95::Tn10 rpoS::kan; from R. Kolter). Only relevant markers are given in parentheses.

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Table 1.

Bacterial strains

Plasmids and phage.pAH85, pSK49, and pSK58 were constructed in an earlier study; each has the same backbone as pSK50ΔuidA2 (13). pAH85 and pSK58 are similar, except that the former has a silent SpeI site at codon 113 of phoB. Each expresses phoB from its native promoter. pSK49 expresses phoB fromPtac . pAH136 and pAH150 are derivatives of pAH85 and pSK49 carrying PrhaB andParaB in place of PphoB and the lacI q-Ptac region, respectively; pAH151 and pAH152 are similar plasmids carryingPrhaB-phoR + andPrhaS-phoR + except that they have the attachment site of HK022 and encode gentamicin resistance (14). pBAD32 and pBAD33 (11) were from L.-M. Guzman. They are similar except that pBAD32 has an undefined deletion of ca. 0.6 kbp between the polylinker and the cat gene of pBAD33 (data not shown). pBC35 is a derivative of pBGS19+ (25) carrying the phoBR operon within a 4.7-kbpPstI-to-EcoRI fragment (32) (Fig.1). pSK47 (16) has the samephoBR fragment cloned into the backbone of pSK50ΔuidA2 (13). pSLF13 and pSLF22 were constructed in an earlier study (8). pSLF13 is a derivative of pSPORT1 (Gibco-BRL, Bethesda, Md.) that encodes a segment of VanS within a 0.4-kbp EcoRI-to-BamHI insert that has a start codon overlapping an NdeI site immediately downstream of a synthetic ribosome-binding site. pSLF22 is a derivative of pBAD32 containing a similar insert. pWJ17 and pWJ18 are derivatives of thelacZ transcriptional fusion vector pWJ13 containing a kanamycin resistance cassette as a PstI fragment, whose loss facilitates recognizing promoter-lacZ fusion plasmids as kanamycin-sensitive (Kans) derivatives (15). Fusions made with these plasmids were recombined onto the chromosome by allele replacement as described below.

Fig. 1.
Fig. 1.

Structure of the phoBR chromosomal region. The 4.7-kbp PstI-to-EcoRI fragment in pBC35 and pSK47 is shown. Asterisks mark the NdeI and BamHI sites that were previously introduced by site-directed mutagenesis upstream and downstream, respectively, of the phoB coding region. The ΔphoB578 mutation was made in pLD82 (Table 2) and recombined onto the chromosome by allele replacement as described in the text. The construction of ΔphoR574 and ΔphoBR580 is described elsewhere (12). Arrows show gene orientations.

All plasmids constructed in this study are described in Table2. Many have the γ replication origin of R6K, denoted oriR R6Kγ, which requires the Π protein (encoded by pir) for replication as a plasmid. Many also contain tetAR, which can be deleterious when present at high copy number. Therefore, these plasmids were routinely maintained in BW23473 or BW24249 or in similarpir + hosts (17). λRZ5lacP-phoU + (PS15) has been described previously (26). Generalized transduction was carried out with P1kc from a laboratory stock.

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Table 2.

Plasmid constructions

Molecular biology methods.PCR amplifications ofara, rha, and phoR sequences were carried out with Vent DNA polymerase (New England Biolabs, Beverly, Mass.) and oligonucleotide primers (Table3; IDT Inc., Coralville, Iowa). Other enzymes were from New England Biolabs or Promega (Madison, Wis.). QIAGEN (Hilden, Germany) products were used for isolation of plasmid DNA, extraction of DNA fragments from agarose gels, and purification of PCR fragments. The phoR, ParaB , andPrhaSB fragments were sequenced on both strands at the Dana Farber Cancer Institute Molecular Biology Core Facility (Harvard Medical School, Boston, Mass.). The ‵araD′ and ‵rhaD fragments (the prime indicates that a gene portion is missing on the side of the prime) were verified only by restriction enzyme analysis.

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Table 3.

Oligonucleotide primers

Molecular genetics.Many new strains were constructed by generalized P1 transduction as described elsewhere (28). BW23321 (Δlac-169 hsdR514 uidA(ΔMluI)::pir + endA BT333) was made by transduction of BW21116 (17) to tetracycline resistance (Tetr) with P1 grown on BT333 followed by transformation of resultant transductant BW23298 with FLP expression plasmid pCP20 and excision of thetetAR genes as described elsewhere (4). The notation endA BT333 signifies the resultant allele. In order to construct strains carrying a minimum number of antibiotic resistance markers, new mutations were usually introduced in two steps. In the first step, a linked auxotrophic marker was introduced by selecting Tetr transductants. These were then made prototrophic with P1 grown on an appropriate donor; the resultant transductants were scored for loss of antibiotic resistance and other relevant phenotypes. Several new mutations and fusions were constructed on plasmids as described above and then recombined onto the chromosome by allele replacement as described elsewhere (17). The resultant chromosomal allele is often given a designation corresponding to the plasmid used in its construction. As necessary, transductants carrying a ΔphoU mutation were verified by complementation with λRZ5lacP-phoU + upon introduction ofphoB + or phoR + or by backcross of the ΔphoU mutation into an appropriate recipient.

The ΔphoB578 mutation (Fig. 1) was recombined onto the chromosome of BW21016 [DE3(lac)X74] by using pLD83 to make BW22901. ThePrhaB-lacZ LD68 andPrhaS-lacZ LD69 fusions were recombined onto the chromosome of BW21578 (rrnB T14 ΔlacZ WJ16) by using pLD68 and pLD69 to make BW22716 and BW22721, respectively. TheParaB-lacZ AH31 fusion was recombined onto the chromosome of BW21480 (lacI q rrnB T14 ΔlacZ WJ16) by using pAH31 to make BW22746. The desired lacZ fusion recombinants were recognized as ones showing a rhamnose- or arabinose-dependent Lac+ phenotype on X-Gal agar. These fusions have the following on the chromosome at the laclocus in a counterclockwise orientation: lacI, four tandem copies of the rrnB transcriptional terminator (denotedrrnB T14), and the foreign promoter preceding thelacZYA operon. The construction of these and similar strains with lacI q rrnB T14 PphnC-lacZ WJ19,lacI q rrnB T14ΔlacZ WJ16, rrnB T14ΔlacZ WJ16, and other promoter-lacZfusions is unpublished (15). Site-specific recombination oforiR R6Kγ attP plasmids onto the chromosome has been described previously (13). All integrants were verified by PCR as described elsewhere (12).

The ParaB and PrhaB fusions were recombined onto the chromosome by using derivatives of new allele replacement plasmids pAH33, pAH54, and pLD78 (Fig.2). This was done with an Ara+ or Rha+ parental strain, so that the resulting segregants with an allele replacement were recognizable as Ara− or Rha− recombinants, respectively. Because these plasmids contain a segment of araD orrhaD, most integrants formed by the initial recombination event are AraD− or RhaD−. Such recombinants are arabinose or rhamnose sensitive, respectively. All selections are therefore carried out in the absence of arabinose or rhamnose.

Fig. 2.
Fig. 2.

Structures of the ParaB andPrhaB allele replacement plasmids. Arrows show gene and promoter orientations. Open boxes in pAH33, pAH54, and pLD78 are promoter regions; hatched boxes are coding regions. All sites are shown for the enzymes indicated. NdeI sites are subscripted to correspond to those indicated in Table 2. pAH33 and pAH54 contain unique PstI and SalI sites downstream ofParaB for construction of aParaB fusion. Genes cloned into these sites require also a ribosome binding site. pLD78 has SalI,XbaI, BamHI, and SphI sites downstream of PrhaB for construction of aPrhaB fusion. pLD78 has the nativerhaB ribosome-binding site upstream of theNdeI2 site, which corresponds to the normalmet start codon of rhaB. Therefore, genes cloned into this site do not require a ribosome-binding site, although partial digestions are required to use this site. An asterisk marksSphI and BglII sites that were lost during cloning of the ‵araD′ fragment. Arrows show gene and promoter orientations. bla, β-lactamase gene;tetA and tetR, tetracycline resistance and repressor genes, respectively; oriT, origin of transfer from RP4; T14 and T1T22, transcription terminators;kan, kanamycin resistance gene; lacZ (op), promoterless lacZ gene for construction of transcriptional, i.e., operon (op), fusions (17). See text.

The ΔaraBAD AH33 mutation was recombined onto the chromosome of BW13711 [DE3(lac)X74] by using pAH33. Integrants carrying pAH33 were selected as Tetr transformants; they were purified once nonselectively, after which Tets segregants were selected on TSS agar. One showing the expected Ara− phenotype was verified and named BW22826. Similarly, theParaB-phoR[M1-D431]AH35 fusion was recombined onto the chromosome of BW21555 (ΔphoR574) by using pAH35 to make BW22835. Recombination of theParaB-phoR[M1-D431]AH35 fusion onto the chromosome results also in removal of the same sequences eliminated by the ΔaraBAD AH33 mutation. Hence, the resultant allele is denoted ΔaraBAD AH33::ParaB-phoR[M1-D431]AH35. The ΔrhaBAD LD78 mutation and ΔrhaBAD LD78::PrhaB-phoB LD79fusion were recombined onto the chromosome of BW22860 [Δ(phoBR brnQ)525 Δcya-161] by using pLD78 and pLD79 to make BW22875 and BW22876, respectively. The desired recombinants were recognized as Rha− ones.

Enzyme assays.β-Galactosidase and bacterial alkaline phosphatase (BAP) assays were carried out as described elsewhere (28).

RESULTS

Construction of allele replacement vectors for tightly regulated protein synthesis from arabinose- and rhamnose-regulated promoters.Since expression of the phoBR operon is subject to positive autogenous control (10), we considered that it would be advantageous to uncouple PhoB synthesis and PhoR synthesis from their normal controls for new studies on the Pho regulon. We therefore constructed a derivative ofParaB plasmid pBAD32 (11) carrying aParaB-phoR + fusion (pAH21; Table 2). However, in preliminary studies, we found that substantial amounts of PhoR were apparently made in the absence of inducer. A ΔphoR mutant carrying pAH21 synthesized BAP upon Pi limitation in the absence of arabinose even under conditions of catabolite repression due to glucose (data not shown). No doubt the “leakiness” of ParaB under these conditions reflects the small amount of PhoR required for activation (phosphorylation) of PhoB. To further reduce PhoR synthesis, we assembled an allele replacement, “suicide” vector system to recombine the ParaB-phoR + fusion onto the chromosome in single copy at the ara locus. We also constructed an analogous allele replacement vector carrying the rhamnose-regulated PrhaB promoter (6) for construction and recombination of aPrhaB-phoB + fusion onto the chromosome in single copy at the rha locus.

Our vectors for constructing chromosomal ParaB and PrhaB fusions are illustrated in Fig. 2. pAH33 and pAH54 are useful for making a ParaB fusion; pLD78 is useful for making a PrhaB fusion. These plasmids contain sequences of the araCBAD orrhaRSBAD locus flanking cloning sites for construction of the respective promoter fusion. pAH33 and pAH54 have a 0.5-kbp fragment containing ParaB and a 0.6-kbp fragment ofaraD upstream and downstream of the cloning region, respectively. They differ in that pAH33 has two NdeI sites (one in tetR and the other in a segment of lacZ) and pAH54 has only one NdeI site (in tetR; Table2). Derivatives of the latter have facilitated the use ofNdeI in particular plasmid constructions (data not shown). pLD78 has a 0.3-kbp fragment containing PrhaB and a 0.6-kbp fragment of rhaD upstream and downstream of its cloning region, respectively. The flanking upstream and downstream sequences provide homologous regions for recombining the fusions onto the chromosome by allele replacement.

pAH33, pAH54, and pLD78 are derivatives of pWJ18 (or pWJ17; Fig. 2), which is in turn a derivative of pir-dependent, counterselectable (tetAR) allele replacement vector pLD53 (17). Upon introduction of these plasmids into a normal (non-pir) Ara+ host (by transformation, electroporation, or conjugation), they cannot replicate and therefore integrate into the chromosome via homologous recombination. Figure3 shows the integration ofParaB-phoR + plasmid pAH35 (Table 2) at the araCBAD locus. Integrants are selectable as Tetr colonies. A subsequent recombination event occurring in the absence of selection leads to the loss of plasmid sequences; this event results in restoration of the parental (wild-type) chromosomal structure or an allele replacement. Recombinants that have lost the plasmid backbone are selectable as Tets ones. Those carrying the desired chromosomal ParaB orPrhaB fusion have the fused gene in single copy at the araCBAD or rhaRSCAB locus in place ofaraBAD or rhaBAD sequences (Fig.4); they are therefore recognizable as Ara− or Rha− ones, respectively. Ones that are also antibiotic sensitive are then verified by genetic linkage or PCR tests. Because the appropriate segregants are Ara− or Rha−, the recombinants provide the additional advantage of not being able to catabolize the respective inducer.

Fig. 3.
Fig. 3.

Recombination of aParaB-phoR + fusion onto the chromosome at the araCBAD locus. The construction of pAH35 is described in Table 2. pAH35 can integrate into the chromosome via either of two homologous recombination events (event A or event B). Only an event A integrant is shown for simplicity. Subsequent recombination events result in loss of the plasmid, regenerating the parental strain (event A) or an allele replacement (event B segregant). These are selectable on TSS agar; ones with an allele replacement are recognizable as Ara− colonies (see Materials and Methods). A ΔphoR mutant was used to recombine theParaB-phoR + fusion onto the chromosome to avoid recombination at the phoBR locus; a wild-type strain can also be used. A black arrow shows the orientation of phoR. Grey arrows show the orientations of other genes. Grey boxes show araC or araD gene fragments and the pir-dependent vector origin (oriR R6Kγ). A prime indicates that a gene portion is missing on the side of the prime. An arrowhead marks the nick site of oriT, the RP4 conjugative transfer origin.

Fig. 4.
Fig. 4.

Allele replacements at the chromosomalaraCBAD and rhaRSBAD regions. (A) Structure of the ΔaraBAD mutant and aParaB-phoR + fusion at thearaCBAD locus. (B) Structure of the ΔrhaBADmutant and a PrhaB-phoB + fusion at the rhaRSBAD locus. Black arrows representParaB-phoR and PrhaB-phoBfusions. Light grey arrows signify genes belonging to theara and rha gene clusters. Dark grey arrows show the yabI and polB genes flanking theara locus and the sodA and yiiL genes flanking the rha locus. Dotted lines indicate regions where homologous recombination between the plasmid and the chromosome can occur. See text.

Measurements of arabinose and rhamnose regulation of single-copy promoter-lacZ fusions.To judge the regulatory capabilities of the arabinose- and rhamnose-regulated promoters in this system, we constructed ParaB -,PrhaB -, and PrhaS-lacZfusions and recombined them onto the chromosome in single copy at thelac locus (Materials and Methods). We then examinedlacZ expression by measuring β-galactosidase levels when strains were grown in the presence and absence of the respective inducer on different carbon sources. Different carbon sources were used because these promoters are subject to catabolite repression. The results are shown in Table 4. In brief, arabinose or rhamnose led to induction of >100-fold to several thousandfold, depending upon the promoter, inducer, and carbon source. As expected, the lowest expression levels were obtained during growth on glucose, for which catabolite repression is the strongest; intermediate expression levels were obtained during growth on fructose, for which catabolite repression is less severe; the highest expression levels were obtained during growth on glycerol, for which catabolite repression is the weakest (7). Differences in both the basal (uninduced) and induced levels exist. In the absence of inducer, the basal level was about 10-fold higher for ParaB than for PrhaB or PrhaS . The induced levels were similar for ParaB andPrhaB (compare BW22831 with BW22887), while the induced levels for PrhaS were ca. 25% of those for PrhaB (compare BW22886 with BW22888).

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Table 4.

Regulation of ParaB -,PrhaB -, andPrhaS-lacZ fusions

In the course of this study, we found that several of our strains carry an rpoS(Am) mutation (Table 1), which is also present in many lines of E. coli K-12, including progenitor K-12 strains EMG2 and W1485 (1, 22). As shown in Table 4, the induced level of PrhaB expression is significantly lower in an rpoS + strain. Apparently, this rpoS effect is related to catabolite repression, as the magnitude varies with the carbon source. The reason for this is unknown. The induction levels in this study are therefore directly comparable only among strains having the same rpoSallele.

Strategy for using ParaB andPrhaB fusions to study regulation of the Pho regulon.We constructed a number of ΔphoB and ΔphoR mutants in which PhoB or PhoR is synthesized under the control of a foreign inducible promoter(s). We did this for two reasons. First, we considered that such strains may be useful in our studies on protein-protein interactions between their gene products, as reported elsewhere (13). Second, we suspected that the deleterious effects of a ΔphoU mutation were in part due to high-level expression of the Pho regulon. Because expression of thephoBR operon is under positive autogenous control, high-level expression of the Pho regulon probably requires also high-level synthesis of PhoB and PhoR. We therefore expected to overcome these deleterious effects by down regulating PhoB or PhoR synthesis by using these foreign-regulated promoters.

Arabinose-independent expression of ParaB fusions.The above results show the ParaB promoter to be tightly regulated for expression of the lacZstructural gene. To assess whether ParaB is useful for tight control of a regulatory gene under similar conditions, we constructed strains carrying chromosomalParaB-phoB + andParaB-phoR + fusions. Each strain has also a precise deletion of the respective regulatory gene. They have in addition a deletion of creC (ΔcreBCD or ΔcreABCD) as well as genes for acetyl phosphate synthesis [Δ(ackA pta)], thus eliminating activation of PhoB by the kinase CreC or acetyl phosphate (33). We then examined PhoB- and PhoR-dependent control by measuring BAP levels under conditions of Pi limitation in the absence of arabinose during growth on different carbon sources. As shown in Table5,ParaB-phoB + ΔphoBstrain BW24803 exhibits a null phenotype in the absence of arabinose on all carbon sources. In contrast,ParaB-phoR + ΔphoRstrain BW22861 exhibits a PhoR+ phenotype in the absence of arabinose, even during growth on glucose. These results are consistent with less PhoR than PhoB being required for transcriptional activation of phoA. Therefore, ParaB appears to be sufficiently tightly controlled for the expression ofphoB, but not for the expression of phoR. Yet,phoR expression is clearly limiting under these conditions, as much higher BAP levels are seen in the presence of arabinose (data not shown).

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Table 5.

Inducer-independent expression ofParaB-phoB + andParaB-phoR + fusions

An eventual goal was to express both phoB andphoR independently and simultaneously from a foreign promoter(s). In order to find appropriate conditions to do this, we used strains with a deletion of the pstSCAB-phoU operon that leads to constitutive expression of the Pho regulon. Individual ones are also ΔphoB or ΔphoR as well as ΔcreC and Δ(ackA pta), as described above. As shown in Table 6, phoAexpression is abolished in the absence of PhoB or PhoR (compare BW24741 and BW24740 with BW24739). Introduction of aPphoB-phoB + fusion in single copy elsewhere on the chromosome restores normal phoA expression (compare BW24770 with BW24739). Introduction of aPtac-phoB + fusion in single copy restores phoA expression partially in the absence of IPTG and fully in the presence of IPTG (compare BW24775 without and with IPTG). This apparent leakiness of Ptac was expected, as proper regulation by LacI requires additional upstream and downstream operator sequences (20) that are absent in thePtac-phoB + fusion. Yet partial inducer dependence is observed even for expression of a regulatory gene from Ptac.

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Table 6.

Inducer-dependent control by using aPtac-phoB + fusion

Arabinose- and rhamnose-dependent expression ofParaB , PrhaB , andPrhaS fusions.We compared strains carrying chromosomalParaB-phoB +,PrhaB-phoB +,ParaB-phoR +,PrhaB-phoR +, andPrhaS-phoR + fusions to determine which promoter fusion(s) and growth conditions were appropriate for studying gene regulation in the Pho regulon. This was done with strains that are otherwise similar to ones described above. We examined PhoB- and PhoR-dependent control of phoA expression by measuring BAP levels when strains were grown on one of four carbon sources (glucose, mannitol, fructose, or glycerol), which were expected to result in different levels of catabolite repression (7). As shown in Table 7, strains carrying aParaB-phoB + orPrhaB-phoB + fusion express a null phenotype in the absence of inducer. The same ones synthesized ca. 12-fold to 2,400-fold more BAP in the presence of inducer, depending upon the carbon source and promoter. The relative expression levels correlate well with expectation in regards to catabolite repression. Catabolite repression is more severe with glucose than mannitol, more severe with mannitol than fructose, and more severe with fructose than glycerol. The BAP levels in theseParaB-phoB + andPrhaB-phoB + fusion strains (ca. 280 to 490 U; BW24774, BW24777, and BW24773) (Table 7) during growth on glycerol are similar to that in an otherwise isogenic wild-type strain (ca. 375 U; BW24739) (Table 6). Strains with aPrhaB-phoR + fusion at two chromosomal sites were examined; no difference between them was seen.

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Table 7.

Inducer-dependent control by usingParaB-phoB +,PrhaB-phoB +,ParaB-phoR +,PrhaB-phoR +, andPrhaS-phoR + fusions

Of the three phoR fusion strains examined, only one (PrhaB-phoR + fusion strain BW24768) showed a null phenotype in the absence of inducer. Substantial activation was apparent in both theParaB-phoR + andPrhaS-phoR + fusion strains (BW24508 and BW24858) in the absence of inducer even during growth on glucose, indicating that both ParaB andPrhaS are somewhat leaky. Their basal levels of expression are also subject to catabolite repression, as they vary with the carbon source. Upon induction with rhamnose, thePrhaB-phoR + fusion strain synthesized ca. 17-fold to 2,300-fold more BAP; these BAP levels also correlate well with the carbon source. The addition of the respective inducer resulted in increased BAP synthesis in theParaB-phoR + andPrhaS-phoR + fusion strains as well.

Use of foreign promoters to bypass growth defect due to a ΔphoU mutation.We showed above that we can modulate expression of the Pho regulon in ourParaB-phoB +,PrhaB-phoB +, andPrhaB-phoR + fusion strains by growing them on different carbon sources in the presence of the respective inducer. We therefore tested whether these conditions can be used to overcome the deleterious effect of a ΔphoUmutation. Whereas a ΔphoU mutant such as BW17142 grows extremely poorly under all conditions tested, an otherwise isogenic Δ(pstSCAB-phoU) mutant such as BW17335 grows reasonably well (Table 8) (26). These strains have a kanamycin resistance cassette in place ofphoU and pstSCAB-phoU sequences, respectively. A similar strain with an unmarked ΔphoU mutation (BW18897; Table 8) also shows a severe growth defect. Yet all these strains synthesize similar amounts of BAP. To determine whether modulating expression of phoB or phoR can be used to overcome the growth inhibition due to a ΔphoU mutation, we constructed otherwise isogenic ΔphoU and Δ(pstSCAB-phoU) strains with a ΔphoB and ΔphoR mutation carrying the respective fusions. These strains have an unmarked ΔphoU mutation, as each has a kanamycin resistance marker elsewhere (Table 1).

View this table:
Table 8.

Pho regulon control in ΔphoU mutants by using ParaB-phoB +,PrhaB-phoB +, andPrhaB-phoR + fusions

As shown in Table 8, our ΔphoU ΔphoB, Δ(pstSCAB-phoU) ΔphoB, ΔphoU ΔphoR, and Δ(pstSCAB-phoU) ΔphoRstrains carrying a ParaB-phoB +,PrhaB-phoB +, orPrhaB-phoR + fusion grow equally well in the absence or presence of the respective inducer. TheParaB-phoB + andPrhaB-phoB + fusion strains (BW24774, BW24950, BW24773, and BW24949) show a null phenotype in the absence of inducer. Each synthesizes ca. 500-fold more BAP in the presence of the respective inducer. Likewise, thePrhaB-phoR + fusion strains (BW24768 and BW24948) show a null phenotype in the absence of rhamnose; each synthesizes ca. 500-fold more BAP in its presence. Upon induction these strains synthesize ca. 25% of the normal amount of BAP (see BW24739 for comparison), suggesting that this level of Pho regulon gene expression is apparently insufficient to result in a severe growth defect due to a ΔphoU mutation.

DISCUSSION

Our primary goal was to develop a method(s) to study PhoU function. The severe growth defect of a ΔphoU mutant is especially problematic due to the rapid accumulation of compensatory mutants with lesions in phoB, phoR, or apst gene. A Pst+ ΔphoU mutant is also exquisitely sensitive to extracellular Pi, suggesting that PhoU has, in addition to its function in Pi signaling, a role as an enzyme in intracellular Pi metabolism (26). As shown here, we were able to overcome the deleterious effect of a ΔphoU mutation by uncoupling PhoB or PhoR synthesis from its normal autogenous control and expressingphoB or phoR from a foreign promoter(s). To do this, we constructed strains carrying aParaB-phoB +,PrhaB-phoB +,ParaB-phoR +, orPrhaB-phoR + fusion on the chromosome. We used strains with single-copy chromosomal fusions to avoid problems resulting from the use of plasmids, such as an antibiotic requirement, variable plasmid copy number, and even plasmid loss. We also examined ways for modulating expression levels from theParaB and PrhaB promoters. We already reported work with similar strains and growth conditions to study the regulatory genes of the Enterococcus faecium vancomycin resistance gene cluster in an E. coli model system (12).

We constructed the corresponding ParaB andPrhaB fusion strains with conditionally replicative (pir-dependent) allele replacement plasmids pAH33, pAH54, and pLD78 (Fig. 2). Strains carrying these fusions are made via a simple two-step procedure as illustrated in Fig. 3. The resulting ParaB and PrhaB fusions reside on the chromosome at the araCBAD andrhaRSBAD loci, respectively, as shown in Fig. 4. We made similar strains with the respective ΔaraBAD AH33 and ΔrhaBAD LD78 chromosomal mutations as controls. In these ways, we constructed a number of strains that express various normal or mutant pho, cre, or vangenes under the control of ParaB orPrhaB in single copy on the chromosome at the respective loci (12-14, 16).

The ParaB promoter (also calledPBAD ) has been shown elsewhere to be tightly regulated (11, 21). In those studies, genes that are normally expressed at moderate levels were examined. In preliminary studies, we found that a pBAD plasmid (11) carryingphoR did not provide sufficiently tight control for our purposes, suggesting that these plasmids are somewhat leaky (14). This is consistent with phoR normally being expressed at a very low level. In order to reduce this basal level of expression, we recombined several ParaB-phoRfusions onto the chromosome by using derivatives ofParaB fusion allele replacement plasmid pAH33. When single-copy fusion strains were used, particularParaB-phoR fusions, e.g., one expressing only the C-terminal kinase domain of PhoR (13), no longer appeared to be leaky. Yet a single-copyParaB-phoR + fusion strain synthesizes sufficient PhoR for (partial) activation in the absence of arabinose even in the presence of glucose (Table 5). These results suggest that full-length PhoR is a more active kinase than its C-terminal domain. However, in the absence of arabinose, the amount of PhoR synthesis from a ParaB-phoR +fusion is also clearly limiting because upon Pi limitation only ca. 10% as much BAP is made in this strain as in a wild-type strain. In contrast, a single-copyParaB-phoB + fusion strain shows no leakiness as it exhibits a PhoB− phenotype in the absence of inducer on all carbon sources tested (Table 7). Therefore, more PhoB than PhoR is apparently required for expression of the Pho regulon. These data are consistent with PhoR acting catalytically as an autokinase and phosphotransferase in the activation (phosphorylation) of PhoB, which in turn acts as a transcription factor.

In order to control PhoB and PhoR synthesis independently, we also constructed strains with aPrhaB-phoB + orPrhaB-phoR + fusion. Although bothl-arabinose and l-rhamnose act directly as inducers for expression of regulons for their catabolism, important differences exist in regard to the regulatory mechanisms (Fig.5). l-Arabinose acts as inducer with the activator AraC in the positive control of the arabinose regulon (23). However, the l-rhamnose regulon is subject to a regulatory cascade; it is therefore subject to an even tighter control. l-Rhamnose acts as an inducer with the activator RhaR for synthesis of RhaS, which in turn acts as an activator in the positive control of the rhamnose regulon (6). As shown in Table 7, both thePrhaB-phoB + andPrhaB-phoR + fusion strains show a PhoB− and PhoR− phenotype, respectively, in the absence of rhamnose. We also examined aPrhaS-phoR + fusion. Like theParaB-phoR + fusion strain, thePrhaS-phoR + fusion strain synthesized a substantial, though limited, amount of BAP in the absence of induction.

Fig. 5.
Fig. 5.

Transcriptional activation of the arabinose and rhamnose regulons. (A) Positive control of the l-arabinose regulon. The araCBAD region near 1.5 min on the E. colichromosome is shown. The unlinked araE and araFGHgenes encode arabinose transport systems (23). (B) Positive control of the l-rhamnose regulon. The rhaRSBADregion near 88.2 min is shown. The nearby rhaT gene is transcribed towards rhaR (Fig. 4) and encodes a rhamnose permease (3). Thick arrows show gene orientations. Curved ones with circled pluses indicate sites for transcriptional activation by AraC (A) or RhaR and RhaS (B). The lower bars show theara and rha sequences in the respectiveParaB and PrhaB fusion allele replacement vectors illustrated in Fig. 2. The dashed lines show the chromosomal regions deleted in the resultant recombinants. See text.

The l-arabinose and l-rhamnose regulons are also regulated by catabolite repression. We therefore modulated expression of ParaB ,PrhaB , and PrhaS fusions by using various carbon sources (glucose, mannitol, fructose, and glycerol) that lead to different levels of catabolite repression (7). When strains are grown on these carbon sources with the inducer in excess, the expression of the Pho regulon can be modulated 70- to 200-fold in our ParaB-phoB +,PrhaB-phoB +, andPrhaB-phoR + fusion strains (Table7). As expected, the differences are much smaller in theParaB-phoR + andPrhaS-phoR + fusion strains as the expression of these fusions is also leaky under these conditions. In preliminary experiments, we had also attempted to modulate expression levels by using different inducer concentrations. However, we were unable to maintain a constant, steady state level of induction (data not shown) (5). An inability to maintain steady-state induction at intermediate levels by the limiting inducer concentration is expected as the presence of arabinose and rhamnose results also in increased synthesis of their respective transport systems (Fig. 5). This has now been substantiated by monitoringParaB expression levels in cells grown in the presence of subsaturating inducer concentrations (24). Importantly, as shown here, expression of these promoters can be modulated over a wide range by using different carbon sources in the presence of saturating inducer concentrations.

Both PhoU and the Pst transporter have been implicated in the negative control of the Pho regulon, as mutations of either result in high constitutive Pho regulon gene expression. Presumably, they somehow act together. To study how they interact with the PhoB-PhoR system, we made strains that synthesize PhoB or PhoR under control of the tightly regulated ParaB and PrhaB promoters. By using these strains, we were able to expressphoB or phoR at a reduced level and thereby overcome the harmful effect of a ΔphoU mutation. In these ways, it should now be possible to study further the function(s) of PhoU.

ACKNOWLEDGMENTS

We thank individuals cited in the text for providing strains and plasmids.

This research is supported by NIH grant GM57695. The Dana Farber Cancer Institute Molecular Biology Core Facility is supported by NIH grants CA06516 and AI28691.

FOOTNOTES

    • Received 30 September 1997.
    • Accepted 2 January 1998.

  • ↵* Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, Lilly Hall of Life Sciences, West Lafayette, IN 47907. Phone: (765) 494-8034. Fax: (765) 494-0876. E-mail: BLW{at}bilbo.bio.purdue.edu.

  • ↵† Present address: Biotechnology Centre, Faculty of Pure and Applied Sciences, University of the West Indies, Mona, Kingston 7, Jamaica.

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