Characterization of PitA and PitB from Escherichia coli

Abstract

ABSTRACT Escherichia coli contains two major systems for transporting inorganic phosphate (P i ). The low-affinity P i transporter ( pitA ) is expressed constitutively 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 pho regulon and is an ABC transporter. We isolated a third putative P i transport 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 containing 500 μM P i , E. coli with wild-type genomic pitB ( pitA Δ pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB Δ pstC345 triple mutant. The mutation Δ pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB + pitA Δ pstC345 strain to utilize P i , with P i uptake rates significantly higher than background levels. In addition, the apparent K m 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 system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.