3. The two-component signal transduction system for Pi homeostasis

Two-component-signaling systems constitute the most common signaling pathways in bacteria [48]. These pathways regulate many important cellular processes ranging from cell development and virulence, to motility and metabolism, and most species have over 10–20 different two-component systems [49]. Most frequently, they are composed of receptors on the cell periphery and signal-processing components and targets in the interior of the cell. These pathways rely on a phospho-transfer reaction between the histidine residues of sensor kinases, which generally receive input from the cell surface, and a conserved aspartate residue within the response regulators, which are located in the cytoplasm [50]. Response regulators are most frequently, but not always, transcription factors that interact with RNA polymerase [51].

## 3.1. PhoB and PhoR—the transcription factor and the histidine kinase

oxyanion. A clue to understanding the mechanisms of Pi transport through the PstSCAB protein comes from sequence similarities between the molybdate, sulfate, and Pi transporters. The most highly conserved sequences within this group are found in a region of the protein that creates a cavity within the membrane-spanning region and a gate that most likely represents the pathway through which the substrate must pass. The published ModABC structure is of the protein in a nucleotide-free conformation and shows 12 transmembrane helices situated in an inward-facing conformation with the gate at the periplasmic surface of the membrane. It has been proposed that PstSCAB, like other transporters in this superfamily, utilizes an alternating access mechanism to transport their substrates in which they alternate between inwardand outward-facing states that are driven by substrate binding, ATP hydrolysis, ADP release, and subsequent ATP binding (see Figure 2) [44]. ATP binding across the PstB dimer interface would be predicted to close the cavity and lead to an outward-facing structure that can receive Pi from the substrate-loaded, periplasmic PstS protein. This event would trigger ATP hydrolysis that would flip the outward-facing transmembrane components to an inward-facing conformation, thereby opening the gate and allowing Pi to gain access to the cytoplasm. The cycle

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The Pst transporter is most highly expressed when environmental Pi levels are low. For this reason, it was assumed that it played its most important role in Pi transport under those conditions. More recently, it has been proposed that it plays the primary role in Pi transport

protein interacts with the PstB protein and slows transport when cytoplasmic Pi concentrations are high.

Figure 2. Model of the mechanism of Pi import through the PstSCAB transporter. Free Pi is bound within the periplasm and presented to the outward-facing PstCAB proteins. This docking triggers ATP hydrolysis, which causes a conformational change that triggers the adoption of an inward-facing conformation. The transported Pi is then released into the cytoplasm, as well as the Pi from the hydrolysis of ATP. The transporter is reset as PstB binds ATP again. The PhoU

would be continued as ADP is released and ATP is rebound.

In E. coli, gene regulation in response to limiting Pi concentrations depends on the function of seven proteins: the two-component regulatory proteins PhoB and PhoR, as well as the Pst transporter, PstSCAB, and an auxiliary protein PhoU [4]. The hub of this signaling pathway consists of the PhoB and PhoR proteins. PhoB is the response regulator that has an Nterminal receiver domain (Pfam: PF00072, response\_reg) and a C-terminal DNA-binding domain (Pfam: PF00486, trans\_reg\_c). This particular domain architecture represents the largest group of response regulators [31]. The receiver domain has a doubly wound α/β-fold with a central five-stranded beta-sheet [52]. This domain contains the site of aspartyl phosphorylation, which in PhoB is Asp53. The receiver domain of PhoB contains the necessary catalytic residues to transfer a phosphoryl group from the phospho-histidine residue of phospho-PhoR [17]. The C-terminal DNA-binding domain has a winged-helix structure [53]. When PhoB becomes phosphorylated, it forms a dimer that binds to DNA sequences, called pho boxes [17, 53–55]. These short sequences are located upstream of Pho regulon genes to recruit RNA polymerase and initiate transcription by remodeling the RNA polymerase holoenzyme-DNA complex [53, 54, 56].

PhoR is a homodimeric, bifunctional histidine autokinase/phospho-PhoB phosphatase. When environmental Pi is limiting, it autophosphorylates on a conserved histidine residue and subsequently donates this phosphoryl group to PhoB, but when Pi is plentiful, it removes the phosphoryl group from phospho-PhoB [57, 58]. PhoR is an integral membrane protein that is not predicted to contain a significant periplasmic domain but does contain a membranespanning region, a cytoplasmic charged region, a Per-ARNT-Sim (PAS) domain (Pfam: PF00989, PAS) [59], and prototypical dimerization/histidine phosphorylation (DHp; Pfam: PF06580, His\_kinase) and catalytic ATP-binding (CA; Pfam: PF02518, HATPase\_c) domains at its C-terminus (see Figure 3) [57]. PAS domains generally function in signal perception activities [60]. Since PhoR does not contain a significant periplasmic sensory domain, it is assumed that its PAS domain senses a cytoplasmic signal that reflects extracellular Pi concentrations, but the nature of the signal is not completely known. The CA domain harbors the enzymatic activity for transferring a phosphoryl group from ATP to the conserved histidine residue of the DHp domain. The DHp domain consists of a four-helix bundle with the conserved phosphoaccepting histidine residue being positioned midway on one face of one of the helices. It has been shown that phosphorylation of PhoR occurs in cis, where the CA domain of one of the monomers phosphorylates the His residue of the same polypeptide chain [61]. The DHp domain also contains all of the residues necessary for phospho-PhoB phosphatase activity [57]. We propose that the control of the opposing kinase and phosphatase activities of PhoR involves the constraint of the CA domains to prevent their access to the DHp domain and simultaneously exposing the residues of the DHp domain that are required for phosphatase function (see Figure 3). If this proposal is correct, then how are the interactions between the different PhoR domains controlled?

3.2. PstSCAB—the sensor of extracellular Pi

model is provided below.

these metals.

3.3. PhoU—the adaptor protein

In addition to its role in Pi transport, the Pst transporter is also required for signal transduction. Because PhoR does not have a periplasmic domain, it has been assumed that this transporter is the ultimate sensor of extracellular Pi [5]. In fact, if any of the Pst proteins are absent, the Pho regulon becomes unregulated, leading to the overexpression of Pho regulon genes [5]. Thus, the default biochemical activity of PhoR is an autokinase and the role of the Pst transporter is to negatively regulate this activity and to stimulate its phospho-PhoB phosphatase activity. There are two possibilities for how the Pst protein may function to control the activity of PhoR. The first is by controlling intracellular Pi levels. If PhoR senses intracellular Pi, most likely through its PAS domain, then the Pst system may function by controlling the amount of Pi within the cell. This model seems unlikely for two reasons. Intracellular Pi has been measured by phosphorous nuclear magnetic resonance (31P NMR) and has been shown to be constant under conditions in which the Pho regulon is both repressed and derepressed [2]. Also, there are several mutations in pstC and pstA that lead to defective transporters, but that retain their signaling capacity, that is, they can still stimulate the phospho-PhoB phosphatase activity of PhoR [40, 41]. The second model for how the Pst transporter functions in signal transduction is that PhoR may somehow sense its transport activity [62]. That is to say, it is not the intracellular level of Pi that is sensed, but how active the transporter is. Support for this

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In addition to the PstSCAB protein, PhoU is also required for Pi-signal transduction, but not for transport through the complex [21]. When phoU is mutated or deleted, PhoR is constitutively active as an autokinase leading to high-level expression of Pho regulon genes. phoU mutants show poor growth and frequently accumulate compensatory mutations in phoR, phoB, or the pstSCAB genes [21, 23, 24]. PhoU is a peripheral membrane protein that modulates Pi transport through the PstSCAB complex [22–24]. When Pi is plentiful, PhoU acts like a brake to prevent too much Pi import, with its accompanying ATP hydrolysis [23]. Multiple crystal structures have been reported for PhoU proteins from various organisms [63–65]. PhoU consists of two symmetric, three alpha-helix bundles and metal ions are found associated with two of these structures. The metals are coordinated by highly conserved amino acid residues that are found in each three-helix bundle. PhoU from Thermotoga maritima coordinates iron clusters [63], while PhoU from Streptococcus pneumoniae shows zinc ions bound [64]. Gardner et al. have recently shown that the soluble form of PhoU from E. coli is a dimer that binds manganese or magnesium [66]. Mutagenesis experiments demonstrated that these divalent metals are bound by the same conserved amino acid residues that bind the iron and zinc ions in the two crystal structures. It was also suggested in this study that metal binding may be important for PhoU interactions with the membrane. Alternatively, PhoU may bind Pi through its interactions with

Two general classes of models have been previously suggested for how PhoU participates in the signaling pathway. It may mediate the formation of a signaling complex between the PstSCAB transporter and PhoR [5, 64] or it may produce a soluble messenger that is

Figure 3. A signaling model involving different conformations of PstSCAB and PhoR. As the Pst transporter switches between its inward- and outward-facing conformations during Pi transport, it interacts differently with PhoR, depending upon its conformation. This interaction is mediated by PhoU. The inward-facing conformation, which is stabilized by the pstBQ160K mutation, interacts with PhoR to constrain its CA domain in order to stabilize the phosphatase conformation of PhoR. The outward conformation, which is stabilized by the pstBE179Q mutation, does not interact with the CA domain and favors the kinase conformation of PhoR, which allows the CA domain to bind ATP and autophosphorylate its DHp domain.

## 3.2. PstSCAB—the sensor of extracellular Pi

PF06580, His\_kinase) and catalytic ATP-binding (CA; Pfam: PF02518, HATPase\_c) domains at its C-terminus (see Figure 3) [57]. PAS domains generally function in signal perception activities [60]. Since PhoR does not contain a significant periplasmic sensory domain, it is assumed that its PAS domain senses a cytoplasmic signal that reflects extracellular Pi concentrations, but the nature of the signal is not completely known. The CA domain harbors the enzymatic activity for transferring a phosphoryl group from ATP to the conserved histidine residue of the DHp domain. The DHp domain consists of a four-helix bundle with the conserved phosphoaccepting histidine residue being positioned midway on one face of one of the helices. It has been shown that phosphorylation of PhoR occurs in cis, where the CA domain of one of the monomers phosphorylates the His residue of the same polypeptide chain [61]. The DHp domain also contains all of the residues necessary for phospho-PhoB phosphatase activity [57]. We propose that the control of the opposing kinase and phosphatase activities of PhoR involves the constraint of the CA domains to prevent their access to the DHp domain and simultaneously exposing the residues of the DHp domain that are required for phosphatase function (see Figure 3). If this proposal is correct, then how are the interactions between the

340 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

Figure 3. A signaling model involving different conformations of PstSCAB and PhoR. As the Pst transporter switches between its inward- and outward-facing conformations during Pi transport, it interacts differently with PhoR, depending upon its conformation. This interaction is mediated by PhoU. The inward-facing conformation, which is stabilized by the pstBQ160K mutation, interacts with PhoR to constrain its CA domain in order to stabilize the phosphatase conformation of PhoR. The outward conformation, which is stabilized by the pstBE179Q mutation, does not interact with the CA domain and favors the kinase conformation of PhoR, which allows the CA domain to bind ATP and autophosphorylate

different PhoR domains controlled?

its DHp domain.

In addition to its role in Pi transport, the Pst transporter is also required for signal transduction. Because PhoR does not have a periplasmic domain, it has been assumed that this transporter is the ultimate sensor of extracellular Pi [5]. In fact, if any of the Pst proteins are absent, the Pho regulon becomes unregulated, leading to the overexpression of Pho regulon genes [5]. Thus, the default biochemical activity of PhoR is an autokinase and the role of the Pst transporter is to negatively regulate this activity and to stimulate its phospho-PhoB phosphatase activity. There are two possibilities for how the Pst protein may function to control the activity of PhoR. The first is by controlling intracellular Pi levels. If PhoR senses intracellular Pi, most likely through its PAS domain, then the Pst system may function by controlling the amount of Pi within the cell. This model seems unlikely for two reasons. Intracellular Pi has been measured by phosphorous nuclear magnetic resonance (31P NMR) and has been shown to be constant under conditions in which the Pho regulon is both repressed and derepressed [2]. Also, there are several mutations in pstC and pstA that lead to defective transporters, but that retain their signaling capacity, that is, they can still stimulate the phospho-PhoB phosphatase activity of PhoR [40, 41]. The second model for how the Pst transporter functions in signal transduction is that PhoR may somehow sense its transport activity [62]. That is to say, it is not the intracellular level of Pi that is sensed, but how active the transporter is. Support for this model is provided below.

### 3.3. PhoU—the adaptor protein

In addition to the PstSCAB protein, PhoU is also required for Pi-signal transduction, but not for transport through the complex [21]. When phoU is mutated or deleted, PhoR is constitutively active as an autokinase leading to high-level expression of Pho regulon genes. phoU mutants show poor growth and frequently accumulate compensatory mutations in phoR, phoB, or the pstSCAB genes [21, 23, 24]. PhoU is a peripheral membrane protein that modulates Pi transport through the PstSCAB complex [22–24]. When Pi is plentiful, PhoU acts like a brake to prevent too much Pi import, with its accompanying ATP hydrolysis [23]. Multiple crystal structures have been reported for PhoU proteins from various organisms [63–65]. PhoU consists of two symmetric, three alpha-helix bundles and metal ions are found associated with two of these structures. The metals are coordinated by highly conserved amino acid residues that are found in each three-helix bundle. PhoU from Thermotoga maritima coordinates iron clusters [63], while PhoU from Streptococcus pneumoniae shows zinc ions bound [64]. Gardner et al. have recently shown that the soluble form of PhoU from E. coli is a dimer that binds manganese or magnesium [66]. Mutagenesis experiments demonstrated that these divalent metals are bound by the same conserved amino acid residues that bind the iron and zinc ions in the two crystal structures. It was also suggested in this study that metal binding may be important for PhoU interactions with the membrane. Alternatively, PhoU may bind Pi through its interactions with these metals.

Two general classes of models have been previously suggested for how PhoU participates in the signaling pathway. It may mediate the formation of a signaling complex between the PstSCAB transporter and PhoR [5, 64] or it may produce a soluble messenger that is recognized by the cytoplasmic domains of PhoR (consistent with observations reported by Hoffer and Tommassen [67] and by Rao and Torriani [68]). The following section presents new evidence in favor of the Pi-signaling complex model.

3.5. Conformational signaling model

phatase activity is stimulated.

in times of Pi sufficiency.

4. The response to high levels of extracellular Pi

To answer the question of how PhoR senses the signaling activity of the Pst transporter, we propose that PhoR interacts differently with the alternate outward- and inward-facing conformations of the transporter that are sampled throughout the transport cycle. When Pi is limiting, the transporters are not actively importing Pi and reside primarily in the outward-facing conformation. We propose that this conformation contacts PhoU in such a manner that it does not interact with both the PAS and CA domains of PhoR, which promotes its autokinase activity. It is only under Pi-replete environments when Pi import is occurring that the Pst transporter adopts the inward-facing conformation. We propose that in this conformation, it interacts with PhoU in such a manner to constrain the CA domain of PhoR so that its phos-

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To test this model, we have recently created two pstB mutations that are predicted to lock the transporter into these alternate conformations (Vuppada and McCleary, manuscript in preparation). Based upon work with the homologous maltose transporter [73], an E179Q mutation in pstB should lock the transporter into an outward-facing conformation because it cannot hydrolyze ATP and a Q160K mutation should lock it into an inward-facing conformation because it does not bind ATP. The pstSCAB-phoU genes were cloned onto a mediumcopy number plasmid and were introduced into a ΔpstSCAB-phoU strain of E. coli to confirm that the plasmid could complement the deletion mutation. Mutations were then introduced into the plasmid by site-directed mutagenesis and confirmed by DNA sequencing. Neither the E179Q nor the Q160K mutants showed high-affinity Pi transport, showing that the transporters were dead. By using alkaline phosphatase expression as a reporter of the Pho regulon, we observed that the E179Q mutant constitutively signaled Pistarvation, whether the cells were grown in Pi-replete or Pi-starvation media. We also observed that the Q160K mutant always signaled Pisufficiency. In other words, these cells always expressed low levels of alkaline phosphatase, presumably resulting from the activation of the phosphatase activity of PhoR. These results support the model in which the inward-facing form of the Pst transporter interacts with PhoU and PhoR in a manner that stimulates the phospho-PhoB phosphatase activity of PhoR. This signaling output of the PhoBR pathway reduces the expression of the PstSCAB transporter when low-level expression is sufficient for maximal growth. It also downregulates other genes whose expression would be wasteful

Clues to understanding how E. coli and other bacteria cope with high levels of extracellular Pi came from studies on Pi remediation [12, 74, 75]. Excess Pi in natural water sources is a major cause of eutrophication [76]. Toxic cyanobacterial blooms are frequently attributed to Pi accumulation in water sources resulting from agricultural runoff [77]. Normally, Pi is removed from wastewaters by chemical precipitation, which is an expensive process [78]. Biological Pi removal is an alternative to chemical treatments in which bacteria accumulate

### 3.4. Protein interactions within the Pi-signaling complex

It has recently been demonstrated through bacterial two-hybrid analysis and through coelution experiments that PhoU interacts with both the PhoR protein and PstB [66]. The twohybrid experiments used the BACTH system [69]. Adenylate cyclase from Bordetella pertussis can be genetically divided into nonfunctional T18 and T25 fragments. The enzyme can be reconstituted in vivo and cAMP can be produced when the two fragments are brought into physical proximity within the cell by fusing interacting proteins with either the T18 or T25 fragments and monitoring cAMP production. Gardner et al. fused various parts of PhoR, or the PstB protein, to the T25 fragment and PhoU to the T18 fragment and indirectly monitored cAMP production by assaying the cAMP-dependent gene β-galactosidase [66]. They found that the interaction between PhoR and PhoU occurred through its PAS domain and that the PstB-PhoU interaction was weaker than the PhoR-PhoU interaction. They then employed a complementary co-elution method by using His-tagged versions of either PstB or PhoR and showed by Western blotting that PhoU was retained on a nickel column in a PstB- or PhoR-dependent manner. In a subsequent paper, Gardner et al. were able to further localize the sites on PhoR and PhoU that are important for the formation of the signaling complex [70]. They knew that the phenotype of a mutant containing the phoU35 allele was unlike that of a phoU deletion mutation. Neither the phoU35 nor the phoU deletion mutants could signal Pi sufficiency and they both constitutively expressed alkaline phosphatase. However, the phoU35 mutant did not have a severe growth defect [71]. Since the phoU35 allele encoded a change from alanine at position 147 to glutamic acid (A147E) [72], Gardner et al. hypothesized that the phoU35 mutation may disrupt PhoU's interaction with PhoR, preventing the signal for the switch to PhoR phosphatase activity, but that it maintained its interaction with PstB, limiting excess transport of Pi into the cell during Pi-replete conditions. From this assumption, they were able to identify the surface residues Ala147 and Arg148 of PhoU as being important for the interaction with PhoR. Moreover, they employed a scanning mutagenesis approach to identify a surface on the PAS domain of PhoR that is essential for the interaction. Every two amino acids within the PAS domain were sequentially mutated and then tested using the BACTH assay for interactions with PhoU. They identified residues 141–146, 157–162, and 169–176 of PhoR as important for the interaction with PhoU. By using these genetic constraints, they were able to build a plausible three-dimensional model of the docked proteins. This model was then supported by using a bioinformatic method, called direct-coupling analysis that identifies residues from one sequence that tend to co-evolve with residues from another sequence. Proteins that physically interact co-evolve with each other. These analyses supported a model in which PhoU interacts with both the PAS and CA domains of PhoR. Gardner et al. proposed the existence of a Pi-signaling complex in which under high-Pi growth conditions PhoU interacts with PhoR to constrain its CA domain and inhibit its kinase activity and promote its phosphatase activity.

### 3.5. Conformational signaling model

recognized by the cytoplasmic domains of PhoR (consistent with observations reported by Hoffer and Tommassen [67] and by Rao and Torriani [68]). The following section presents new

It has recently been demonstrated through bacterial two-hybrid analysis and through coelution experiments that PhoU interacts with both the PhoR protein and PstB [66]. The twohybrid experiments used the BACTH system [69]. Adenylate cyclase from Bordetella pertussis can be genetically divided into nonfunctional T18 and T25 fragments. The enzyme can be reconstituted in vivo and cAMP can be produced when the two fragments are brought into physical proximity within the cell by fusing interacting proteins with either the T18 or T25 fragments and monitoring cAMP production. Gardner et al. fused various parts of PhoR, or the PstB protein, to the T25 fragment and PhoU to the T18 fragment and indirectly monitored cAMP production by assaying the cAMP-dependent gene β-galactosidase [66]. They found that the interaction between PhoR and PhoU occurred through its PAS domain and that the PstB-PhoU interaction was weaker than the PhoR-PhoU interaction. They then employed a complementary co-elution method by using His-tagged versions of either PstB or PhoR and showed by Western blotting that PhoU was retained on a nickel column in a PstB- or PhoR-dependent manner. In a subsequent paper, Gardner et al. were able to further localize the sites on PhoR and PhoU that are important for the formation of the signaling complex [70]. They knew that the phenotype of a mutant containing the phoU35 allele was unlike that of a phoU deletion mutation. Neither the phoU35 nor the phoU deletion mutants could signal Pi sufficiency and they both constitutively expressed alkaline phosphatase. However, the phoU35 mutant did not have a severe growth defect [71]. Since the phoU35 allele encoded a change from alanine at position 147 to glutamic acid (A147E) [72], Gardner et al. hypothesized that the phoU35 mutation may disrupt PhoU's interaction with PhoR, preventing the signal for the switch to PhoR phosphatase activity, but that it maintained its interaction with PstB, limiting excess transport of Pi into the cell during Pi-replete conditions. From this assumption, they were able to identify the surface residues Ala147 and Arg148 of PhoU as being important for the interaction with PhoR. Moreover, they employed a scanning mutagenesis approach to identify a surface on the PAS domain of PhoR that is essential for the interaction. Every two amino acids within the PAS domain were sequentially mutated and then tested using the BACTH assay for interactions with PhoU. They identified residues 141–146, 157–162, and 169–176 of PhoR as important for the interaction with PhoU. By using these genetic constraints, they were able to build a plausible three-dimensional model of the docked proteins. This model was then supported by using a bioinformatic method, called direct-coupling analysis that identifies residues from one sequence that tend to co-evolve with residues from another sequence. Proteins that physically interact co-evolve with each other. These analyses supported a model in which PhoU interacts with both the PAS and CA domains of PhoR. Gardner et al. proposed the existence of a Pi-signaling complex in which under high-Pi growth conditions PhoU interacts with PhoR to constrain its CA domain and inhibit its kinase activity and promote its

evidence in favor of the Pi-signaling complex model.

phosphatase activity.

3.4. Protein interactions within the Pi-signaling complex

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To answer the question of how PhoR senses the signaling activity of the Pst transporter, we propose that PhoR interacts differently with the alternate outward- and inward-facing conformations of the transporter that are sampled throughout the transport cycle. When Pi is limiting, the transporters are not actively importing Pi and reside primarily in the outward-facing conformation. We propose that this conformation contacts PhoU in such a manner that it does not interact with both the PAS and CA domains of PhoR, which promotes its autokinase activity. It is only under Pi-replete environments when Pi import is occurring that the Pst transporter adopts the inward-facing conformation. We propose that in this conformation, it interacts with PhoU in such a manner to constrain the CA domain of PhoR so that its phosphatase activity is stimulated.

To test this model, we have recently created two pstB mutations that are predicted to lock the transporter into these alternate conformations (Vuppada and McCleary, manuscript in preparation). Based upon work with the homologous maltose transporter [73], an E179Q mutation in pstB should lock the transporter into an outward-facing conformation because it cannot hydrolyze ATP and a Q160K mutation should lock it into an inward-facing conformation because it does not bind ATP. The pstSCAB-phoU genes were cloned onto a mediumcopy number plasmid and were introduced into a ΔpstSCAB-phoU strain of E. coli to confirm that the plasmid could complement the deletion mutation. Mutations were then introduced into the plasmid by site-directed mutagenesis and confirmed by DNA sequencing. Neither the E179Q nor the Q160K mutants showed high-affinity Pi transport, showing that the transporters were dead. By using alkaline phosphatase expression as a reporter of the Pho regulon, we observed that the E179Q mutant constitutively signaled Pistarvation, whether the cells were grown in Pi-replete or Pi-starvation media. We also observed that the Q160K mutant always signaled Pisufficiency. In other words, these cells always expressed low levels of alkaline phosphatase, presumably resulting from the activation of the phosphatase activity of PhoR. These results support the model in which the inward-facing form of the Pst transporter interacts with PhoU and PhoR in a manner that stimulates the phospho-PhoB phosphatase activity of PhoR. This signaling output of the PhoBR pathway reduces the expression of the PstSCAB transporter when low-level expression is sufficient for maximal growth. It also downregulates other genes whose expression would be wasteful in times of Pi sufficiency.
