1. Introduction

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 serine, threonine, and tyrosine.

The presence of the PhoBR signal transduction system underscores the need for maintaining a minimal intracellular level of Pi when extracellular Pi is limiting. That too much intracellular Pi can also be a problem is underscored by the phenotype of a phoU mutant [21]. phoU is the fifth gene in the pstSCAB-phoU operon and its function is to control the activity and the amount of the PstSCAB transporter [22]. It has been shown that phoU mutations cause a severe growth defect, probably because these cells become poisoned by too much intracellular Pi [21, 23, 24]. Taken together, these observations suggest that E. coli cells possess homeostatic mechanisms that maintain intracellular Pi levels within an optimal range. It is the purpose of this chapter to introduce the reader to the principle players involved in Pi homeostasis and to highlight

Molecular Mechanisms of Phosphate Homeostasis in *Escherichia coli*

http://dx.doi.org/10.5772/67283

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E. 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

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,

contained compensatory mutations in other genes that restored growth on Pi [21].

advances in our understanding of the mechanisms involved.

2. The multiple Pi importers

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

potential binding partners, or means of regulation.

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 amount of polyphosphate, a Pi storage compound.

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 upand down-regulated.

The presence of the PhoBR signal transduction system underscores the need for maintaining a minimal intracellular level of Pi when extracellular Pi is limiting. That too much intracellular Pi can also be a problem is underscored by the phenotype of a phoU mutant [21]. phoU is the fifth gene in the pstSCAB-phoU operon and its function is to control the activity and the amount of the PstSCAB transporter [22]. It has been shown that phoU mutations cause a severe growth defect, probably because these cells become poisoned by too much intracellular Pi [21, 23, 24]. Taken together, these observations suggest that E. coli cells possess homeostatic mechanisms that maintain intracellular Pi levels within an optimal range. It is the purpose of this chapter to introduce the reader to the principle players involved in Pi homeostasis and to highlight advances in our understanding of the mechanisms involved.
