Journal of Bacteriology, September 2001, p. 5008-5014, Vol. 183, No. 17
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5008-5014.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
School of Biochemistry and Molecular Biology, The Faculties,1 and Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research,2 The Australian National University, ACT, 0200, Australia
Received 2 May 2001/Accepted 11 June 2001
Escherichia coli contains two major systems for
transporting inorganic phosphate (Pi). The low-affinity
Pi 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 Pi concentrations by the pho
regulon and is an ABC transporter. We isolated a third putative
Pi transport gene, pitB, from E.
coli K-12 and present evidence that pitB encodes a functional Pi transporter that may be repressed at low
Pi levels by the pho regulon. While a
pitB+ cosmid clone allowed growth on medium
containing 500 µM Pi, 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 Pi, with
Pi 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 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.
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