2. The multiple Pi importers

1. Introduction

serine, threonine, and tyrosine.

and down-regulated.

the amount of polyphosphate, a Pi storage compound.

Inorganic phosphate (Pi) is essential for life. For example, it is found in the hydrophilic ends of the amphipathic lipids in the cellular membranes that define the boundaries of a cell. Together with the sugars ribose or deoxyribose, it makes up the structural backbone of DNA or RNA through its phosphodiester bonds. The cell's energy currency is based upon the energy released from the hydrolysis of the phosphoanhydride bonds between the phosphates of ATP or of the other nucleotides. Moreover, the biochemical activities of many proteins are regulated by the phosphorylation of specific amino acids—histidine and aspartate in bacteria, as well as

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Because of its essential roles, cells must maintain intracellular Pi pools at optimal levels. In bacteria, such as Escherichia coli, this is believed to be between 1 and 10 mM [1–3]. Pi is assimilated into biological molecules through the synthesis of ATP from ADP and Pi. The mechanisms to control intracellular Pi levels include multiple transport proteins with characteristic patterns of expression, different affinities for Pi, and rates of Pi transport [4]. E. coli cells also employ a well-studied sensory transduction system that monitors extracellular Pi levels to control the expression of genes for scavenging Pi under limiting conditions and to utilize alternate phosphorous sources. Additionally, there are also metabolic reactions that control

The primary Pi importers in E. coli are PitA, PitB, and PstSCAB [5]. PitA and PitB are secondary transporters that bring neutral metal-Pi complexes into the cell at the expense of a proton [6, 7]. PstSCAB is a Pi-specific ABC transporter that imports Pi at the expense of ATP hydrolysis [8, 9]. Proteins that export Pi include PitA, PitB, and GlpT, which is a glycerol-3-phosphate:Pi antiporter [10], UhpT, which is a hexose-6-phosphate:Pi antiporter [11], and potentially YjbB, which has been suggested to be a Pi exporter [12]. The signal transduction system that controls gene expression in response to limiting extracellular Pi levels has at its heart the histidine kinase PhoR and the response regulator PhoB [4, 13]. When PhoB receives a phosphoryl group from PhoR, it binds to DNA and activates the transcription of a number of genes for the high-affinity acquisition of Pi (including the PstSCAB transporter) and the utilization of alternate sources of phosphorous [14–17]. At least 31 genes have been shown to be directly controlled and positively regulated by PhoB. They are called the Pho regulon and include phoA, which encodes the periplasmic enzyme alkaline phosphatase, pstSCAB, phoB, and phoR [4]. Alkaline phosphatase removes phosphoryl groups from organophosphate molecules. The members of the Pho regulon that are involved in utilizing alternate phosphorous sources are ugpBAECQ, which encodes a glycerol-3-phosphate ABC transporter and a phosphodiesterase and phnCDEFGHIJKLMNOP, which encodes a phosphonate transporter and enzymes of a C-P lyase complex that produces a phosphoribosyl product from imported phosphonate. Phosphonates are compounds that contain a carbon-phosphorous bond. In addition to the 31 genes that have been demonstrated to be directly controlled by PhoB [4, 18], 2Dpolyacrylamide gels and computational methods suggest that possibly 400 proteins may be controlled directly or indirectly by PhoB [19, 20]. These include genes that are both upE. coli is capable of using multiple transporters to bring Pi into cells. Three of them, PitA, PitB, and PstSCAB, are individually capable of supporting growth with Pi as the only source of phosphorous [6]. The others, GlpT, UhpT, and PhnCDE, are capable of secondarily importing Pi but are not able to support growth when the sole source of phosphorous is Pi [5]. GlpT primarily transports glycerol-3 phosphate, UhpT transports hexose-6-phosphates and PhnCDE brings phosphonates into the cell. Complicating many of the early studies on Pi transport was the use of the K10 strain of E. coli, which harbored a G220D mutation in the pitA gene [25]. The interpretations of some of the genetic and biochemical studies of Pi transport in these strains are therefore difficult because many early strains contained compensatory mutations in other genes that restored growth on Pi [21].

### 2.1. PitA and PitB—the low-affinity Pi importers

The low-affinity PitA and PitB transporters utilize the energy stored in the proton-motive force to bring neutral metal-Pi complexes into the cell [6, 25, 26]. These homologous proteins each contain 499 amino acid residues and show 80.8 and 89.8% sequence identity and similarity, respectively (see Figure 1A). Amino acid identities between the two proteins are indicated by vertical lines and similarities are indicated with two dots. A membrane topology model for these two proteins was created using the SCAMPI2 web server [27] and is shown in Figure 1B. This model predicts that PitA and PitB have 10 transmembrane helices with the N- and C-termini facing the periplasm. The sequences of the predicted transmembrane helices are surrounded by green boxes in Figure 1A. Support for this Nout-Cout topology model comes from a recent paper in which the authors tagged the C-termini of 601 inner membrane proteins from E. coli with alkaline phosphatase and green fluorescent proteins (GFPs) [28]. Because alkaline phosphatase is only active in the periplasm and GFP is only fluorescent in the cytoplasm, they concluded that PitA and PitB have a Cout topology [28]. These two proteins show very high levels of amino acid identity and similarity within the predicted 10 transmembrane segments (91.4 and 96.7%, respectively). The greatest degree of divergence is found in a putative 127-amino acid cytoplasmic loop domain (L7) located between helices 7 and 8. This loop shows 59.1% identity and 75.6% similarity between the two proteins suggesting that it may contribute to differences in protein stability, potential binding partners, or means of regulation.

function may be more important during growth in Pi-replete environments. The mechanisms

PitA and PitB are members of the PiT family of Pi transporters within the transporter classification database (TC #2.A.20) [30] and the PHO4 family within the Pfam database [31]. These families include bacterial, archaeal, and eukaryotic members, suggesting that these Pi transporters developed early in evolution and that they continue to play important functions in all domains of life. A conserved signature sequence has been identified in both the N- and Cterminal ends of these transporters that has the common core sequence of G(AFGST)(NH) (DN)(VATIG)(AQSG)(NKA)(ASTG)(IMVF)(GAS)(TPIL), with the bolded amino acids representing the most common amino acids at that position. This signature sequence is highlighted with red letters in Figure 1A. The human proteins from this family are thought to be involved in housekeeping functions and are called PiT1 and PiT2, whereas the Neurospora crassa and Saccharomyces cerevisiae members are called Pho-4 and Pho89, respectively [32, 33]. Mutations in the signature sequence of the PiT2 protein block Pi transport [34]. In addition to their role in Pi transport, the PiT1 and PiT2 proteins are also receptors for the gamma-retrovi-

PiT1, PiT2, Pho-4, and Pho89 are sodium-dependent transporters, whereas PitA, PitB, and the

It has recently been suggested that neither PitA nor PitB play primary roles in Pi transport, but function instead for the purpose of metal ion transport [4]. However, considering the homologies between PitA and PitB with other Pi transporters from other organisms, it seems unlikely that they are retained in this genome primarily to function as transporters of divalent metal cations, which have their own primary transporters, as well [36]. Clearly, further work is

The PstSCAB protein is a high-affinity Pi transporter that has a Km of 0.4 μM Pi and a Vmax of 16 nmol Pi mg (dry weight)−<sup>1</sup> min−<sup>1</sup> [37]. It is a member of the ATP-binding cassette (ABC) superfamily from the transporter classification and Pfam databases [30, 31]. This protein superfamily employs the hydrolysis of ATP to bring a variety of substrates across biological membranes, both as importers and as exporters [38]. Members of this protein superfamily are found among the bacteria, archaea, and eukaryotes. Prokaryotic importers, such as the PstSCAB protein, utilize an extra-cytoplasmic substrate-binding protein that binds substrates and presents them to their membrane-spanning proteins [39]. PstS is the periplasmic substratebinding protein. PstC and PstA compose the membrane-spanning components of the transporter [40, 41]. The most highly conserved feature within the superfamily is the nucleotidebinding domain, also called the ATP-binding cassette, which binds ATP, hydrolyzes it, and then releases it in order to provide the energy for transport [42]. PstB contains the nucleotidebinding domain for this transporter [43]. The crystal structures of several ABC importers have been solved, which has shed some light onto the mechanisms of transport [44]. Of particular note is the structure of the putative molybdate transporter, called ModABC, from the archaeon Archaeoglobus fulgidus [45]. Like the PstSCAB transporter, this protein also imports an

Pht2\_1 proteins from Arabidopsis thaliana are proton-dependent Pi symporter [35].

needed to better understand the roles of PitA and PitB in Pi homeostasis.


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for the regulation of these genes are not known.

ruses [32]. This protein family includes both Na+

2.2. PstSCAB—the high-affinity Pi importer

Figure 1. Sequence alignments of PitA and PitB with an accompanying topology model. (A) PitA and PitB amino acid sequences are given in one-letter code and are aligned. The alignment was made using the European Molecular Biology Open Software Suite (EMBOSS) [29]. Predicted transmembrane helices are boxed in green and the conserved signature motifs are marked with a red font. (B) Topology model of PitA and PitB. The model includes an Nout-Cout topology. The predicted transmembrane helices are labeled TM1–TM10 and the connecting loops are labeled L1–L9.

Analysis of the kinetic properties of Pi uptake in whole cells where pitA and pitB were expressed from the pBR322 plasmid showed that the PitA protein has a Km app of 1.9 μM and a Vmaxapp of 58 nmol of Pi minute−<sup>1</sup> milligram (dry weight)−<sup>1</sup> , whereas the values for PitB are 6 μM and 67 nanomoles of Pi minute−<sup>1</sup> milligram (dry weight)−<sup>1</sup> [6].

It was originally thought that pitA expression was constitutive, but it has recently been shown that it is positively regulated by the availability of Zn(II) and also by limiting Pi [7]. pitB expression appears to be repressed when cells are grown in limiting Pi conditions [25], so its function may be more important during growth in Pi-replete environments. The mechanisms for the regulation of these genes are not known.

PitA and PitB are members of the PiT family of Pi transporters within the transporter classification database (TC #2.A.20) [30] and the PHO4 family within the Pfam database [31]. These families include bacterial, archaeal, and eukaryotic members, suggesting that these Pi transporters developed early in evolution and that they continue to play important functions in all domains of life. A conserved signature sequence has been identified in both the N- and Cterminal ends of these transporters that has the common core sequence of G(AFGST)(NH) (DN)(VATIG)(AQSG)(NKA)(ASTG)(IMVF)(GAS)(TPIL), with the bolded amino acids representing the most common amino acids at that position. This signature sequence is highlighted with red letters in Figure 1A. The human proteins from this family are thought to be involved in housekeeping functions and are called PiT1 and PiT2, whereas the Neurospora crassa and Saccharomyces cerevisiae members are called Pho-4 and Pho89, respectively [32, 33]. Mutations in the signature sequence of the PiT2 protein block Pi transport [34]. In addition to their role in Pi transport, the PiT1 and PiT2 proteins are also receptors for the gamma-retroviruses [32]. This protein family includes both Na+ -dependent and H+ -dependent Pi symporters. PiT1, PiT2, Pho-4, and Pho89 are sodium-dependent transporters, whereas PitA, PitB, and the Pht2\_1 proteins from Arabidopsis thaliana are proton-dependent Pi symporter [35].

It has recently been suggested that neither PitA nor PitB play primary roles in Pi transport, but function instead for the purpose of metal ion transport [4]. However, considering the homologies between PitA and PitB with other Pi transporters from other organisms, it seems unlikely that they are retained in this genome primarily to function as transporters of divalent metal cations, which have their own primary transporters, as well [36]. Clearly, further work is needed to better understand the roles of PitA and PitB in Pi homeostasis.

## 2.2. PstSCAB—the high-affinity Pi importer

Analysis of the kinetic properties of Pi uptake in whole cells where pitA and pitB were

Figure 1. Sequence alignments of PitA and PitB with an accompanying topology model. (A) PitA and PitB amino acid sequences are given in one-letter code and are aligned. The alignment was made using the European Molecular Biology Open Software Suite (EMBOSS) [29]. Predicted transmembrane helices are boxed in green and the conserved signature motifs are marked with a red font. (B) Topology model of PitA and PitB. The model includes an Nout-Cout topology. The

It was originally thought that pitA expression was constitutive, but it has recently been shown that it is positively regulated by the availability of Zn(II) and also by limiting Pi [7]. pitB expression appears to be repressed when cells are grown in limiting Pi conditions [25], so its

app of 1.9 μM and

, whereas the values for PitB are 6

expressed from the pBR322 plasmid showed that the PitA protein has a Km

predicted transmembrane helices are labeled TM1–TM10 and the connecting loops are labeled L1–L9.

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a Vmaxapp of 58 nmol of Pi minute−<sup>1</sup> milligram (dry weight)−<sup>1</sup>

μM and 67 nanomoles of Pi minute−<sup>1</sup> milligram (dry weight)−<sup>1</sup> [6].

The PstSCAB protein is a high-affinity Pi transporter that has a Km of 0.4 μM Pi and a Vmax of 16 nmol Pi mg (dry weight)−<sup>1</sup> min−<sup>1</sup> [37]. It is a member of the ATP-binding cassette (ABC) superfamily from the transporter classification and Pfam databases [30, 31]. This protein superfamily employs the hydrolysis of ATP to bring a variety of substrates across biological membranes, both as importers and as exporters [38]. Members of this protein superfamily are found among the bacteria, archaea, and eukaryotes. Prokaryotic importers, such as the PstSCAB protein, utilize an extra-cytoplasmic substrate-binding protein that binds substrates and presents them to their membrane-spanning proteins [39]. PstS is the periplasmic substratebinding protein. PstC and PstA compose the membrane-spanning components of the transporter [40, 41]. The most highly conserved feature within the superfamily is the nucleotidebinding domain, also called the ATP-binding cassette, which binds ATP, hydrolyzes it, and then releases it in order to provide the energy for transport [42]. PstB contains the nucleotidebinding domain for this transporter [43]. The crystal structures of several ABC importers have been solved, which has shed some light onto the mechanisms of transport [44]. Of particular note is the structure of the putative molybdate transporter, called ModABC, from the archaeon Archaeoglobus fulgidus [45]. Like the PstSCAB transporter, this protein also imports an 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 would be continued as ADP is released and ATP is rebound.

under all conditions [4]. The expression of the pstSCAB genes is controlled by the PhoBR twocomponent system described below. The primary promoter for this operon, and the one which is regulated by Pi levels, is found upstream of the pstS gene [46]. Other promoters that are internal to the operon have been identified upstream of the pstC, pstB, and phoU genes and are rather weak; but they may play a role in maintaining a basal level of the PstSCAB transporter

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

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

merase holoenzyme-DNA complex [53, 54, 56].

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].

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 poly-

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:

under Pi-replete conditions [47].

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

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 under all conditions [4]. The expression of the pstSCAB genes is controlled by the PhoBR twocomponent system described below. The primary promoter for this operon, and the one which is regulated by Pi levels, is found upstream of the pstS gene [46]. Other promoters that are internal to the operon have been identified upstream of the pstC, pstB, and phoU genes and are rather weak; but they may play a role in maintaining a basal level of the PstSCAB transporter under Pi-replete conditions [47].
