Characterization of PitA and PitB from Escherichia coli

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 highaffinity 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 M P i , E. coli with wild-type genomic pitB (pitA ⌬pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB ⌬pstC345 triple mutant. The mutation ⌬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 ⌬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.Escherichia coli contains at least two major systems for transporting inorganic phosphate (P i ). The low-affinity inorganic phosphate transporter (Pit) is dependent on the proton motive force for energy and is constitutively expressed (30,31,49). When P i is plentiful, this is the major uptake system for phosphate, with a reported apparent K m (K m app ) of 25 M (30) to 38 M (50) in whole cells and 11.9 M in membrane vesicles (43). If the external P i concentration is below the millimolar range, the high-affinity phosphate-specific transport (Pst) system is induced. This has a K m app of around 0.2 M (30, 50). The Pst system is a complex of four proteins, including a periplasmic binding protein, which is energized by ATP and belongs to the ABC transporter family (7,15,47). The pst operon contains five genes under pho regulon control (1,40,41), which induces a range of genes when the phosphate supply is limited. Both the Pit and Pst systems are highly specific for P i (30). Another two transporters accept P i as a low-affinity analogue for either glycerol-3-phosphate (glpT) (18) or glucose-6-phosphate (uhpT) (29, 53), but in the absence of Pit and Pst activity, these latter two systems cannot support cell growth when supplied with P i (38).Divalent cations, such as Mg 2ϩ or Ca 2ϩ

Phosphate Transport Kinetics and Structure-Function Relationships of SLC34 and SLC20 Proteins

“…However, one distinguishing feature concerns the driving cation. In prokaryotes and plants, P i transport mediated by these proteins is coupled to the H + gradient (van Veen 1997;Daram, Brunner et al 1999;Harris, Webb et al 2001), whereas in animals and fungi, transport is coupled to the Na + gradient (Versaw&Metzenberg 1995;Martinez&Persson 1998;Tatsumi, Segawa et al 1998;Uckert, Willimsky et al 1998;Bai, Collins et al 2000). SLC20 proteins are presumed to have the same secondary structure (Fig1B, inset) with a common inverted repeat topology that differs significantly from that of SLC34 proteins.…”

Molecular analysis of phosphate limitation in Geobacteraceae during the bioremediation of a uranium-contaminated aquifer

“…For this reason, most bacteria have two phosphate transport systems, which differ in affinity and specificity for phosphate (Bardin and Finan, 1998). The lowaffinity phosphate transport system (Pit) transports phosphate into the cells in the presence of high levels of extracellular phosphate (Gachter and Meyer, 1993;Voegele et al, 1997;Bardin and Finan, 1998;Van Dien and Keasling, 1999;Botero et al, 2000;Harris et al, 2001). It is usually constitutively expressed and is dependent on the proton motive force.…”

Investigation of the properties of wild type and mutant alkaline phosphatase variants: A laboratory project linking genotype and phenotype

“…2). This does not correspond exactly to what the mutants actually are but was done to give the students a somewhat simpler system to analyze because the pho regulon is quite complex [20]. In particular, AN4085 is used for both M4 and M5.…”

“…These strains are ampicillin resistant. For week 3, strains AN248 (wild type), AN2537 (constitutively active phoA), and AN4085 (phoA not expressed) were used [20]. These strains are not ampicillin resistant.…”