Characterization of PitA and PitB from Escherichia coli

Escherichia coli contains two major systems for transporting inorganic phos phate (P-i). The low-affinity P-i transporter (pitA) is expressed constitut ively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external P-i concentrations by the p ho regulon and is an ABC transporter. We isolated a third putative PI trans port gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P-i transporter that may be repressed at low P-i levels by the pho regulon. While a pitB(+) cosmid clone allowed growth on medium containi ng 500 muM P E. coli with wild-type genomic pitB (pitA Delta pstC345 double mutant) was unable to grow under these conditions, making it indistinguish able from a pitA pitB Delta pstC345 triple mutant. The mutation Delta pstC3 45 constitutively activates the pho regulon, which is normally induced by p hosphate starvation. Removal of pho regulation by deleting the phoB-phoR op eron allowed the pitB(+) pitA Delta pstC345 strain to utilize P-i, with P-i uptake rates significantly higher than background levels. In addition, the apparent Km of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst syst em is mutated. The pitA mutation was identified as a single base change, ca using an aspartic acid to replace glycine 220. This mutation greatly decrea sed the amount of PitA protein present in cell membranes, indicating that t he aspartic acid substitution disrupts protein structure.