Employment of a Promoter-Swapping Technique Shows that PhoU Modulates the Activity of the PstSCAB 2 ABC Transporter in Escherichia coli

Abstract

ABSTRACT Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (P i ). Under these conditions, the high-affinity PstSCAB 2 protein (i.e., with two PstB proteins) is the primary P i transporter. Expression from the pstSCAB-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 of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the P tac promoter and the lacO ID regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli . Here we utilized P phoB :: P tac and P pstS :: P tac strains to characterize phenotypes resulting from various Δ phoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB 2 transporter, as well as its abundance within the cell. In addition, we used the P phoB :: P tac Δ phoU strain as a platform to begin characterizing new phoU mutations in plasmids.