Open AccessEmployment of a Promoter-Swapping Technique Shows that PhoU Modulates the Activity of the PstSCAB 2 ABC Transporter in Escherichia coli
Author(s) -
Christopher Rice,
Jacob Pollard,
Zachery T. Lewis,
William R. McCleary
Publication year - 2009
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.01046-08
Subject(s) - escherichia coli , escherichia coli proteins , atp binding cassette transporter , enterobacteriaceae , transporter , genetics , biology , computational biology , microbiology and biotechnology , gene
Expression of the Pho regulon inEscherichia coli is induced in response to low levels of environmental phosphate (Pi ). Under these conditions, the high-affinity PstSCAB2 protein (i.e., with two PstB proteins) is the primary Pi transporter. Expression from thepstSCAB-phoU operon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies ofphoU have proven to be difficult because deletion of thephoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability ofphoU deletions, we employed a promoter-swapping technique that places expression of thephoBR two-component system under control of thePtac promoter and thelacO ID regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes inE. coli . Here we utilizedPphoB ::Ptac andPpstS ::Ptac strains to characterize phenotypes resulting from various ΔphoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB2 transporter, as well as its abundance within the cell. In addition, we used thePphoB ::Ptac ΔphoU strain as a platform to begin characterizing newphoU mutations in plasmids.