Part:BBa_K4252009

Phosphate-specific transportor (Pst)

The phosphate (Pi)-specific transport system of Escherichia coli (Pst) is a typical ABC transport system composed of four different proteins: PstS, the periplasmic Pi-binding protein; PstC and PstA, integral membrane proteins that mediate the translocation of Pi through the inner membrane and PstB that binds ATP and energizes the transport. The operon that encodes Pst contains five genes in the following order: pstS, pstC, pstA, pstB, and a fifth distal gene, phoU, whose product does not play a role in the transport of Pi. The Pst system encodes an ATPbinding cassette (ABC) transporter involved in the transport of inorganic phosphate (Pi). As a member of the PHO regulon, the Pst operon is induced in response to Pi limitation. In order to allow E. coli to absorb more Pi, we link the Pst (only contains pstS, pstC, pstA, pstB) gene onto the expression vector pET-28a(+) and introduced it into E.coli BL21. Through inducing its expression by IPTG, E. coli BL21 can efficiently absorb Pi when the external Pi isn’t in limitation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 154
  Illegal AgeI site found at 720
  Illegal AgeI site found at 2721
  Illegal AgeI site found at 3917
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 3737
  

Introduction

Phosphorous, in the form of phosphate, is a key element in the nutrition of all living beings. In nature, it is present in the form of phosphate salts, organophosphates, and phosphonates. Echerichia coli transport inorganic phosphate by two different routes. The first one is a low-affinity transport system, Pit system (phosphate inorganic transport system), which is expressed constitutively and is dependent on the proton motive force, catalyzes a rapid transport process between both sides of phosphate pools. The other way is called Pst system (phosphate-specific transport system). Research has revealed that the high-affinity Pst system (pstSCAB) is induced at low external Pi concentrations by the pho regulon and is an ABC (ATP-binding cassette) transporter, which means Pst system will still work when the concentration of external Pi is lower than 20 microM instead of Pit system. Whereas the high affinity of Pst and its character of inducible expression, our team decide to transfer pstSCAB into Escherichia coli. The Pst system consists of four components, in order of PstS, PstC, PstA, PstB, and other regulator genes such as phoB, phoR and phoU are used to control the inoriginic phosphate transport and others like phoA, phoE, phoP to control the other forms of phosphorus.

• PstS:

PiBP, a protein which is determined by PstS gene, is also a phosphate-binding protein and discriminates between arsenate and phosphate, which attached to the outer side of the cell membrane and it could combine with phosphate in periplasmic space and transport it to the membrane (also an ABC-transporter).

• PstA and PstC:

PstA and PstC determine pstA and pstC and both of which are hydrophobic protein and they form the transmembrane portion of the Pst system.

• PstB:

PstB determine pstB protein that is the catalytic subunit and interact on the cytoplasmic side, which couples the energy of ATP hydrolysis to control the open and close of phosphate channel by the alpha-helix domains of PstA and PstC. And phosphate molecule can across the channel by the salt bridge composed of Arg and Glu.

• Regulator (PhoB, PhoR, PhoU) :

The Pho regulon is controlled by a two-component regulatory system which comprises an inner-membrane histidine kinase sensor protein and a cytoplasmic transcriptional response regulator. The system in E.coli is named PhoB-PhoR. The PhoB encodes a positive transcriptional activator, phoB, and PhoR encodes a phosphate-sensory protein, phoR. The PhoB is already a dimer before binding to the DNA, but it has to be phosphorylated in order to become active: it bind a upstream from genes of the Pho regulon, which is directly for the PstS. The PhoR protein has a dual regulatory role as both an activator and a repressor. Under Pi limitation, PhoB is activated by PhoR acting as a kinase, but under Pi-replete conditions, PhoB activation is interrupted by PhoR acting as a phosphatase. PhoU is required for PhoB dephosphorylation under Pi-rich conditions in an unknown way. When phoU is mutated or deleted, PhoR behaves as a constitutive PhoB kinase, leading to high expression of the Pho regulon genes. PhoU is involved not only in control of the autokinase activity of PhoR, but also in the control of the Pst system to avoid an uncontrolled Pi uptake that could be toxic for the cell.

Figure:The regulation of Pst.